Tetra-substituted ndga derivatives via ether bonds and carbamate bonds and their synthesis and pharmaceutical use

ABSTRACT

Disclosed are nordihydroguaiarctic acid derivative compounds including various end groups bonded by a carbon atom or heteroatom though a side chain bonded to the respective hydroxy residue O groups by an ether bond or a carbamate bond, pharmaceutical compositions, methods of making them, and methods of using them and kits including them for the treatment of diseases and disorders, in particular, diseases resulting from or associated with a virus infection, such as HIV infection, HPV infection, or HSV infection, an inflammatory disease, such as various types of arthritis and inflammatory bowel diseases, a metabolic disease, such as diabetes, a vascular disease, such as hypertension and macular degeneration, or a proliferative disease, such as diverse types of cancers.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of Ser. No. 13/470,779, filed May 14,2012, now U.S. Pat. No. 9,067,875, which is a Continuation of Ser. No.12/443,906, filed Jun. 5, 2009, now U.S. Pat. No. 8,178,527, which is aU.S. National Phase application under 35 U.S.C. §371 of InternationalPatent Application No. PCT/US2007/080182, filed Oct. 2, 2007, whichclaims the benefit of U.S. Provisional Patent Application No.60/827,776, filed Oct. 2, 2006, which applications are incorporatedherein by reference in their entireties.

BACKGROUND OF THE INVENTION

The present invention relates to nordihydroguaiaretic acid derivatives,methods of making them and methods of using them for treating viralinfections, inflammatory diseases, metabolic diseases, vascular(including cardiovascular) diseases and proliferative diseases, such ascancer.

Nordihydroguaiaretic acid (NDGA, Formula I) has the following chemicalstructure, in which there are two catechol groups, and a2,3-dimethylbutane bridge. The butane bridge links two catechol moietiesthrough a 4 position. NDGA is a natural compound that can be isolatedfrom the resin of the leaves of Larrea tridentata, a desert plantindigenous to the southwestern United States and Mexico. It has ameso-form conformation of (2S,3R), which is the symmetric structure, andis not optical active.

Research on NDGA and its derivatives has been attracting increasinginterest recently. A large number of NDGA derivatives have beenreported, and could be classified as the following:

Type 1: ether bonded NDGA, the most common NDGA derivatives, in which asubstituted group is chemically bonded to one or more of the hydroxygroups of the catechol moieties.

Type 2: ester bonded NDGA derivatives, in which a substituted group iscovalently bonded to one or more of the hydroxy groups of the catecholmoieties.

Type 3: end-ring NDGA derivatives, in which two hydroxy groups at thecatechol moieties were linked together to form 5-6 member rings throughether or carbonate bonds.

Type 4: di-substituted NDGA derivatives, in which one hydroxy group ofthe catechol is methylated, the other one is covalently bonded to asubstituted group.

Type 5: phenyl ring modifications, in which the substituted groups arechemically linked to the phenyl ring.

Type 6: Butane bridge modifications, in which two methyl groups in thebridge were removed or modified by substituted groups.

NDGA and its synthetic derivatives have numerous characteristics. Beinga lipoxygenase inhibitor. NDGA can induce cystic nephropathy in therat.¹ In addition, it shows various bioactivities, including inhibitionof protein kinase C,² induction of apoptosis,³ alterations of thecellular membrane,⁴ elevation of cellular Ca²⁺ level⁵ and activation ofCa²⁺ channels in smooth muscle cells,⁶ breakdown of pre-formedAlzheimer's beta-amyloid fibrils in vitro,⁷ anti-oxidation,⁸ etc. Thisnatural product NDGA is used commercially as a food additive to preservefats and butter in some parts of the world. Recently, the derivatives ofthe plant lignan NDGA have been used to block viral replication throughthe inhibition of viral transcription.⁹⁻¹⁶ These compounds can inhibitproduction of human immunodeficiency virus (HIV),⁹⁻¹³ herpes simplexvirus (HSV),^(14, 15) and human papillomavirus (HPV) transcripts¹⁶ bydeactivation of their Sp1-dependent promoters. Moreover,(tetra-O-methyl)nordihydroguaiarctic acid (M₄N, Formula II,terameprocol) can function as an anti-HIV proviral transcriptioninhibitor and causes growth arrest of a variety of transformed human andmouse cells in culture and in mice.¹⁷⁻¹⁹ Compound M₄N is currently inclinical trials against human cancers.

While M₄N (Formula II) is a strikingly effective and non-toxicanticancer agent, M₄N and several other methylated NDGAs, all show poorwater solubility which somewhat limit their application for certain drugaction studies. To circumvent this problem, a water soluble derivativeof NDGA, (tetra-O-dimethylglycyl)nordihydroguaiaretic acid (G₄N, FormulaIII) has been designed and synthesized.^(11, 18)

G₄N is a very effective mutation-insensitive inhibitor to HIV-1, HSV-1and HSV-2.¹⁷ However, it is somewhat unstable and has a relatively shorthalf-life in aqueous solution, reportedly due to the ester bondsconnecting the dimethyl glycine moieties on the NDGA main skeleton.⁸

Therefore, there is a need for NDGA derivatives, some with improvedwater solubility, as well as good stability, both as water solublecompound and as hydrophobic compounds having the desired pharmaceuticaleffects. The inventors have developed new derivatives of NDGA that havethese advantages and will be useful in therapeutic compositions andtreatment methods.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to nordihydroguaiaretic acid derivativecompounds, pharmaceutical compositions, methods of making them, andmethods of using them and kits including them for the treatment ofdiseases and disorders, in particular, diseases resulting from orassociated with a virus infection, an inflammatory disease, a metabolicdisease, a vascular disease or a proliferative disease.

One aspect of the present invention relates to a nordihydroguaiareticacid derivative compound designated “Sb₄N”, having the following generalstructure (Formula IV), as well as its pharmaceutically acceptablesalts:

The compound has a nordihydroguaiaretic acid (NDGA) backbone, designatedby “N” in the designation “Sb₄N.” The four groups X are substituted forH in the NDGA hydroxyl groups (sometimes referred to as the “substitutedgroup X”) designated by “Sb₄” in “Sb₄N.”

X is selected from the group consisting of:

-   -   -A-R;    -   —(CH₂)_(x)Hal, where x is an integer of 1 to 10, and Hal is a        halogen atom, namely any of chlorine, fluorine, bromine or        iodine;    -   —(CH₂CH₂O)_(y)H, where y is an integer of 1 to 10; and    -   a carbamate-bonded group selected from the group consisting of:

where n is an integer of 1 to 6, Z₁ is a saturated linear hydrocarbonchain of 2-6 carbons and optionally 1-3 halogen atoms, Z₂ is a 5- to7-member ring optionally containing 0-3 double bonds and optionallycontaining 1-3 atoms of any of O, N and S, and Z₃ is methyl or ethyl.

When X is -A-R, R is an end group and A is a linear saturatedhydrocarbon side chain with optional heteroatoms that is bonded at oneend to the respective hydroxy residue 0 groups by an ether bond or acarbamate bond and at the other end to a carbon or a heteroatom in theend group R.

The side chain A is selected from the group consisting of a C₂-C₁ linearsaturated hydrocarbon chain, optionally with 1-5 heteroatoms selectedfrom the group consisting of O, N and S, bonded to the respectivehydroxy residue O groups of NGDA through an ether bond; and 1-5 units ofa polyethylene glycol (PEG) chain.

The end group R is selected from the group consisting of:

a 5- to 7-member carbocyclic ring selected from the group consisting ofa fully saturated ring with 1 to 3 N, O or S heteroatoms; a ringcontaining 1 to 3 double bonds for a 6- or 7-member ring and 1 to 2double bonds for a 5-member ring, with 1 to 3 N, O or S heteroatoms forthe 5 to 7 member ring; a ring containing a carbamate bond, a urea bond,a carbonate bond or an amide bond; and

a water soluble group selected from the group consisting of an alkalimetal salt of sulfonic acid; an alkali metal salt of phosphonic acid; apharmaceutically acceptable salt; a sugar and a polyhydroxy group.

Where X is

a is an integer of 3 to 16 and b is an integer of 4 to 16.

Another aspect of the invention is a composition comprising the Sb₄Ncompound and a pharmaceutically acceptable carrier, optionally withother pharmaceutically acceptable excipients.

Still another aspect of the invention is a method of making the Sb₄Ncompound as set forth hereinafter.

Another aspect of the invention is a method of administering to asubject, an amount of the Sb₄N compound alone or as part of apharmaceutical composition effective prophylactically or for treating aviral infection.

Yet another aspect of the invention is a method of administering to asubject an amount of the Sb₄N compound alone or as part of apharmaceutical composition effective prophylactically or for treating aproliferative disease.

Another aspect of the invention is a method of administering to asubject an amount of the Sb₄N compound alone or as part of apharmaceutical composition effective prophylactically or for treating aninflammatory disease.

Yet another aspect of the invention is a method of administering to asubject an amount of the Sb₄N compound alone or as part of apharmaceutical composition effective prophylactically or for treating ametabolic disease.

Yet another aspect of the invention is a method of administering to asubject an amount of the Sb₄N compound alone or as part of apharmaceutical composition effective prophylactically or for treating avascular disease.

Still another aspect of the present invention is a kit comprising apharmaceutical composition comprising the Sb₄N compound and instructionsfor its use prophylactically or for treating a viral infection, aproliferative disease, an inflammatory disease, a metabolic disease or avascular disease.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, the present invention relates to nordihydroguaiareticacid derivative compounds, pharmaceutical compositions containing them,methods of making them, and methods of using them and kits includingthem for the treatment of diseases and disorders, in particular, viralinfections, such as, for example and without limitation, infectionscaused by human immunodeficiency virus (HIV), human papillomaviruses(HPV)(all subtypes), herpes simplex virus 1 and 2 (HSV-1 and HSV-2),Varicella Zoster virus, cytomegalovirus, Epstein Barr virus, pox viruses(smallpox, cowpox, monkeypox, vaccinia), orthohepadnavirus, JC virus,and BK virus; inflammatory diseases, such as, for example and withoutlimitation, various types of arthritis and inflammatory bowel diseases;metabolic diseases, such as, for example and without limitation,diabetes; vascular diseases, such as for example hypertension,cardiovascular diseases and macular degeneration; and proliferativediseases such as various types of cancers.

The present invention is based on considerations including experiencewith agents used for treating cancer and viruses, including HIV;derivatives having a chemical structure related to NDGA; where suchderivatives have more potency, better PD/PK profile and less or no sideeffects versus (tetra-O-methyl)nordihydroguaiaretic acid (M₄N)(terameprocol), at least some formulations of which are orallybioavailable. The NDGA derivatives of the present invention are in themeso form without any possible mixtures of their enantiomers, which willmake the chemical and biological characterization easier. Thesubstituent functional groups for the modifications of the NDGA parent,compound are selected from among the most common chemical groups usedfor successful drug molecule modifications. They are readily able to besynthesized and readily formulated with reasonable aqueous solubility,in that in the HCl or other salt form or in free base, they haveconsiderable aqueous solubility. Other of the NDGA derivatives of thepresent invention are hydrophobic. The NDGA derivatives of the presentinvention have good stability, whether they are water soluble compoundsor hydrophobic compounds. The derivatives may be scaled-up readily forcommercial production.

The NDGA derivatives of the present invention were developed based onthe fact that NDGA is natural compound with a broad range of biologicalactivities. NDGA has a lot of side effects, which are overcome by thederivatives of the invention. The derivatives have improved biologicalactivities. The research leading to the development of the presentinvention has also shown that hydroxy group modification of NDGAderivatives, such as M₄N, prevents tumor cell replication andselectively induces tumor cell death (apoptosis). This is achieved bypreventing Sp1-regulated production of cdc2 (p34) and survivin. Survivinis an inhibitor of apoptosis protein (IAP) over-expressed inpre-cancerous and cancerous cells, and rarely found in healthy adultcells. M₄N also prevents proliferation of human immunodeficiency virus(HIV), herpes simplex virus (HSV), and human papilloma virus (HPV). Thisis achieved through the deactivation of viral Sp1-dependent promotersthat are essential for viral propagation. NDGA derivatives of thepresent invention will remarkably improve their activities to preventSp1-regulated production of cdc2 and survivin by using a suitablefunctional group to modify the hydroxyl group of NDGA.

DEFINITIONS

A “buffer” suitable for use herein includes any buffer conventional inthe art, such as, for example, Tris, phosphate, imidazole, andbicarbonate.

A “cyclodextrin” as used herein means an unmodified cyclodextrin or amodified cyclodextrin, and includes without limitation α-cyclodextrin,β-cyclodextrin, γ-cyclodextrin and any modified cyclodextrins containingmodifications thereto, such as hydroxypropyl-f-cyclodextrin (“HP-β-CD”)or sulfobutyl ether β-cyclodextrin (“SBE-β-CD”). Cyclodextrin typicallyhas 6 (α-cyclodextrin), 7 (β-cyclodextrin), and 8 (γ-cyclodextrin)sugars, up to three substitutions per sugar, and 0 to 24 primarysubstitutions are therefore possible (primary substitutions are definedas substitutions connected directly to the cyclodextrin ring). Themodified or unmodified cyclodextrins used in the present invention mayhave any appropriate number and location of primary substitutions orother modifications.

An “NDGA derivative” of the present invention as used herein means aderivative of NDGA designated as “Sb₄N” hereinafter.

A “pharmaceutically acceptable carrier” refers to a non-toxic solid,semisolid or liquid filler, diluent, encapsulating material orformulation auxiliary of any conventional type. A “pharmaceuticallyacceptable carrier” is non-toxic to recipients at the dosages andconcentrations employed, and is compatible with other ingredients of theformulation. For example, the carrier for a formulation containing thepresent catecholic butane or NDGA derivatives preferably does notinclude oxidizing agents and other compounds that are known to bedeleterious to such derivatives. Suitable carriers include, but are notlimited to, water, dextrose, glycerol, saline, ethanol, buffer, dimethylsulfoxide, Cremaphor EL, and combinations thereof. The carrier maycontain additional agents such as wetting or emulsifying agents, or pHbuffering agents. Other materials such as anti-oxidants, humectants,viscosity stabilizers, and similar agents may be added as necessary.

A “pharmaceutically acceptable salt” as used herein includes the acidaddition salts (formed with the free amino groups of the polypeptide)and which are formed with inorganic acids such as, for example,hydrochloric or phosphoric acids, or such organic acids as acetic,mandelic and oxalic acids. Salts formed with the free carboxyl groupsmay also be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, 2-ethylamino ethanol, andhistidine. Non-limiting examples of pharmaceutically acceptable salts ofthe NDGA derivatives of the present invention include, for instance, thefollowing general formula of salts:

[Sb₄N]·k[acid],

where N is NDGA, Sb is a substituted group X as described in Tables 1and 2 below, k is an integer or non integer number, and acid is organicor inorganic acid, as exemplified in the following non-limiting Table A:

TABLE A Sb Acid k Containing one HCl, HBr, HNO₃, MeSO₃H, 1-4 basicnitrogen H₂SO₄, aspartic acid, citric acid, benzenesulfonic atom acid,camphoric acid, camphorsulfonic acid, eth- anesulfonic acid,2-hydroxy-ethansulfonic acid, formic acid, fumaric acid, galactaricacid, D- gluconic acid, glycolic acid, hippuric acid, L- lactic acid,maleic acid, malic acid, malonic acid, nicotinic acid, palmitic acid,pamoic acid, phosphoric acid, salicylic acid, succinic acid, tartaricacid, p-toluenesulfonic acid. Containing two HCl, HBr, HNO₃, MeSO₃H, 1-8basic nitrogen H₂SO₄, aspartic acid, citric acid, benzenesulfonic atomsacid, camphoric acid, camphorsulfonic acid, eth- anesulfonic acid,2-hydroxy-ethansulfonic acid, formic acid, fumaric acid, galactaricacid, D- gluconic acid, glycolic acid, hippuric acid, L- lactic acid,maleic acid, malic acid, malonic acid, nicotinic acid, palmitic acid,pamoic acid, phosphoric acid, salicylic acid, succinic acid, tartaricacid, p-toluenesulfonic acid.

The term “pharmaceutically acceptable excipient” as used herein includesvehicles, adjuvants, or diluents or other auxiliary substances, such asthose conventional in the art, which are readily available to thepublic. For example, pharmaceutically acceptable auxiliary substancesinclude pH adjusting and buffering agents, tonicity adjusting agents,stabilizers, wetting agents and the like.

A “ring” as used herein, unless otherwise specified, as used herein suchas the terms “5-member ring,” “6-member ring” and “7-member ring,”refers to a carbocyclic ring with any indicated heteroatoms.

The terms “subject,” “host,” and “patient,” are used interchangeablyherein to refer to an animal being treated with the presentcompositions, including, but not limited to, simiens, humans, felines,canines, equines, bovines, porcines, ovines, caprines, mammalian farmanimals, mammalian sport animals, and mammalian pets.

A “substantially purified” compound in reference to the NDGA derivativesherein is one that is substantially free of compounds that are not theNDGA derivative of the present invention (hereafter, “non-NDGAderivative materials”). By substantially free is meant at least 50%,preferably at least 70%, more preferably at least 80%, and even morepreferably at least 90% free of non-NDGA derivative materials.

As used herein, the terms “treatment.” “treating,” and the like, referto obtaining a desired pharmacologic and/or physiologic effect. Theeffect may be prophylactic in terms of completely or partiallypreventing a condition or disease or symptom thereof and/or may betherapeutic in terms of a partial or complete cure for a condition ordisease and/or adverse affect attributable to the condition or disease.“Treatment.” thus, for example, covers any treatment of a condition ordisease in an animal, preferably in a mammal, and more preferably in ahuman, and includes: (a) preventing the condition or disease fromoccurring in a subject which may be predisposed to the condition ordisease but has not yet been diagnosed as having it; (b) inhibiting thecondition or disease, such as, arresting its development; and (c)relieving, alleviating or ameliorating the condition or disease, suchas, for example, causing regression of the condition or disease.

This invention is described by way of example only and is not to beinterpreted in any way as limiting the invention. Thus, this inventionis not limited to particular embodiments described, as such may, ofcourse, vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only, andis not intended to be limiting, since the scope of the present inventionwill be limited only by the appended claims in a non-provisionalapplication based on this provisional application.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

It must be noted that as used herein, the singular forms “a”, “an”, and“the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “a derivative” includes aplurality of such derivatives and reference to “the NDGA derivative”includes reference to one or more NDGA derivatives and equivalentsthereof known to those skilled in the art in view of the presentdisclosure.

All publications mentioned herein, including patents, patentapplications, and journal articles are incorporated herein by referencein their entireties including the references cited therein, which arealso incorporated herein by reference. The publications discussed hereinare provided solely for their disclosure prior to the filing date of thepresent application. Nothing herein is to be construed as an admissionthat the present invention is not entitled to antedate such publicationby virtue of prior invention. Further, the dates of publication providedmay be different from the actual publication dates which may need to beindependently confirmed.

As noted above, one aspect of the present invention relates to an Sb₄Nnordihydroguaiaretic acid derivative compound having the followinggeneral structure (Formula IV), as well as its pharmaceuticallyacceptable salts:

The compound has a nordihydroguaiartic acid (NDGA) backbone, designatedby “N” in the designation “Sb₄N.” The four groups X are substituted forH in the NDGA hydroxyl groups (sometimes referred to as the “substitutedgroup X”) designated by “Sb₄” in “Sb₄N.”

X is selected from the group consisting of:

-   -   -A-R;    -   —(CH₂)_(x)Hal, where x is an integer of 1 to 10, and Hal is a        halogen atom, namely any of chlorine, fluorine, bromine or        iodine;    -   —(CH₂CH₂)_(y)H, where y is an integer of 1 to 10; and a        carbamate-bonded group selected from the group consisting of:

where n is an integer of 1 to 6, Z₁ is a saturated linear hydrocarbonchain of 2-6 carbons and optionally 1-3 halogen atoms, Z₂ is a 5- to7-member ring optionally containing 0-3 double bonds and optionallycontaining 1-3 atoms of any of O, N and S, and Z₃ is methyl or ethyl.

Where X is —(CH₂)_(x)Hal, x is preferably 1 to 3, and Hal is chlorine orfluorine; more preferably, in this instance, X is —(CH₂)₂F, for example.

Where X is —(CH₂CH₂CH)_(y)H, y is preferably 1 to 3, and morepreferably, for example, in this instance, X is —(CH₂)₂OH (when y is 1);or —(CH₂)₂—O—(CH₂)₂—OH (when y is 2).

One proviso is where X is

a is an integer of 3 to 16 and b is and integer of 4 to 16.

With the foregoing additional definitions of X and the proviso, the sidechain A and the end groups R may be selected from those set forth in theBrief Summary of the Invention above, wherein the side chain A and theend groups R are set forth in tabular form in the following Table 1:

TABLE 1 Side chain A C₂-C₁₆ linear saturated hydrocarbon chainoptionally with 1-5 N, O or S heteroatoms, and the chain is bonded atone end to the respective hydroxy groups residue O of NGDA through anether bond and at the other end to a carbon or heteroatom of the endgroup R 1-5 units of polyethylene glycol (PEG) chain R is a 7- fullysaturated 7-member ring with 1 to 3 N, O or member ring S heteroatoms7-member ring containing 1 to 3 double bonds with 1 to 3 N, O or Sheteroatoms 7-member ring containing a carbamate bond, a urea bond, acarbonate bond or an amide bond R is a 6- fully saturated 6-member ringwith 1 to 3 N, O or member ring S heteroatoms 6-member ring containing 1to 3 double bonds with 1 to 3 N, O or S heteroatoms 6-member ringcontaining a carbamate bond, a urea bond, a carbonate bond or an amidebond R is a 5- fully saturated 5-member ring with 1 to 3 N, O or memberring S heteroatoms 5-member ring containing 1 to 2 double bonds with 1to 3 N, O or S heteroatoms 5-member ring containing a carbamate bond, aurea bond, a carbonate bond or an amide bond R is a water an alkalimetal salt of sulfonic acid soluble group an alkali metal salt ofphosphonic acid a pharmaceutically acceptable salt, such as shown inTable A a sugar a polyhydroxy group

Non-limiting examples of a suitable side chain A are C₂-C₄ linear chain,such as ethylene, propylene or butylene, bonded at one end to therespective hydroxy residue 0 groups of NDGA through an ether bond; C₂-C₄linear chain, such as ethylene, propylene or butylene, with an O or Nheteroatom, and the chain is bonded to the respective hydroxy residue Ogroups of NGDA through an ether bond; 1-3 units of polyethylene glycol(PEG) chain; or a carbamate bond. The side chain is bonded at the otherend to a carbon or heteroatom of the end group R.

Non-limiting examples of suitable end groups R are set forth in thefollowing Table 2:

TABLE 2 R is 5-7 member carbocyclic ring containing 1-3 N, O or Sheteroatoms

R is polyhydroxy, sugar, or other water soluble group, where m is aninteger of 2 to 6

General methods to synthesize the ether bonded NDGA derivatives are asfollows:

General Method 1: Reaction of Alkyl Halide with NDGA Under BasicCatalytic Conditions:

General Method 2: Reaction of Toluenesulfonic Acid Activated Alcoholwith NDGA:

General methods to synthesize the carbamate bonded NDGA derivatives areas follows:

General Method 1: Reaction of an Isocyanate Compound with NDGA

General Method 2: Reaction of N-Succinimidyl N-Substituted Carbamatewith NDGA

Details of the preparation of exemplary specific NDGA derivativecompounds according to the present invention will be set forth below inthe Examples section.

The present NDGA derivatives in a suitable formulation, preferably butnot exclusively as the active ingredient or as one of two or more activeingredients in a pharmaceutical composition with a pharmaceuticallyacceptable carrier or excipient where appropriate, can be safelyadministered to a subject in need of such treatment by intranasaldelivery, by inhalation, intravenously such as by infusion or byinjection into the central vein for example, intra-arterially (with orwithout occlusion), intraperitoneally, interstitially, subcutaneously,transdermally, intradermally, intraocularly, intramuscularly, topically,intracranially, intraventricularly, orally, or buccally, or byimplantation.

Moreover, the NDGA derivatives can be safely administered to a subjectin need of such treatment in solution, suspension, semisolid or solidforms as appropriate, or in liposomal formulations, nanoparticleformulations, or micellar formulations for administration via one ormore routes mentioned above.

Furthermore, the NDGA derivatives in liposomal formulations,nanoparticles formulations, or micellar formulations can be embedded ina biodegradable polymer formulation and safely administered, such as bysubcutaneous implantation.

Compositions for administration herein may, be in any suitable form,such as and without limitation, a solution, suspension, tablet, pill,capsule, sustained release formulation or powder, a liquid that iseither hydrophilic or hydrophobic, a powder such as one resulting fromlyophilization, an aerosol, an aqueous or water-soluble composition, ahydrophobic composition, a liposomal composition, a micellar compositionsuch as that based on Tween®80 or diblock copolymers, a nanoparticlecomposition, a polymer composition, a cyclodextrin complex composition,an emulsion, or as lipid based nanoparticles termed “lipocores.”

The present invention further encompasses compositions, includingpharmaceutical compositions, comprising the NDGA derivatives andpharmaceutically acceptable carriers or excipients. These compositionsmay include a buffer, which is selected according to the desired use ofthe NDGA derivatives, and may also include other substances appropriatefor the intended use. Those skilled in the art can readily select anappropriate buffer, a wide variety of which are known in the art,suitable for an intended use, in view of the present disclosure. In someinstances, the composition can comprise a pharmaceutically acceptableexcipient, a variety of which are known in the art. Pharmaceuticallyacceptable excipients suitable for use herein are described in a varietyof publications, including, for example, Gennaro (Gennaro, A.,Remington: The Science and Practice of Pharmacy, 19th edition,Lippincott, Williams, & Wilkins, (1995)); Ansel, et al. (Ansel, H. C. etal., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7^(th)edition, Lippincott, Williams, & Wilkins (1999)); and Kibbe (Kibbe, A.H., Handbook of Pharmaceutical Excipients, 3^(rd) edition Amer.Pharmaceutical Assoc.).

The compositions herein are formulated in accordance to the mode ofpotential administration. Thus, if the composition is intended to beadministered intranasally or by inhalation, for example, the compositionmay be a converted to a powder or aerosol form, as conventional in theart, for such purposes. Other formulations, such as for oral orparenteral delivery, are also used as conventional in the art.

Compositions or formulations suitable for oral or injectable deliveryadditionally includes a pharmaceutical composition containing acatecholic butane for treatment of the indicated diseases where thecomposition is formulated with a pharmaceutically acceptable carrier andother optional excipients, wherein the carrier comprises at least one ofa solubilizing agent and an excipient selected from the group consistingof (a) a water-soluble organic solvent; (b) a ionic, non-ionic oramphipathic surfactant, (d) a modified cellulose; (e) a water-insolublelipid; and a combination of any of the carriers (a)-(e).

The water-soluble organic solvent may be preferably, but notnecessarily, other than dimethyl sulfoxide. Non-limiting exemplarywater-soluble organic insolvents include polyethylene glycol (“PEG”),for example, PEG 300, PEG 400 or PEG 400 monolaurate, propylene glycol(“PG”), polyvinyl pyrrolidone (“PVP”), ethanol, benzyl alcohol ordimethylacetamide.

The cyclodextrin or modified cyclodextrin may be, without limitation,α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, HP-β-CD or SBE-β-CD.

The ionic, non-ionic or amphipathic surfactant may include, for examplewithout limitation, a surfactant such as polyoxyethylene sorbitanmonolaurate (also known as polysorbate); which is a non-ionicsurfactant, for example, polysorbate 20 and polysorbate 80, commerciallyavailable as Tween® 20 or Tween® 80, d-alpha-tocopheryl polyethyleneglycol 1000 succinate (“TPGS”), glycerol monooleate (also known asglyceryl monooleate), an esterified fatty acid or a reaction productbetween ethylene oxide and castor oil in a molar ratio of 35:1,commercially available as Cremophor® EL. Preferably, for certainembodiments, when the surfactant is a non-ionic surfactant, thenon-ionic surfactant is present in the absence of xanthan gum.

Non-limiting examples of a modified cellulose include ethyl cellulose(“EC”), hydroxylpropyl methylcellulose (“HPMC”), methylcellulose (“MC”)or carboxy methylcellulose (“CMC”). In one embodiment of the invention,the catecholic butane may be solubilized in modified celluloses that canbe diluted in ethanol (“EtOH”) prior to use.

The water-insoluble lipids include, for example, an oil or oils, such ascastor oil, sesame oil or peppermint oil, a wax or waxes, such asbeeswax or carnuba wax, and mixed fat emulsion compositions such asIntralipid® (Pharmacia & Upjohn, now Pfizer), used as per themanufacturer's recommendation. For example, adult dosage is recommendedto be not exceeding 2 g of fat/kg body weight/day (20 mL, 10 mL and 6.7mL/kg of Intralipid® 10%, 20% and 30%, respectively). Intralipid® 10% isbelieved to contain in 1,000 mL: purified soybean oil 100 g, purifiedegg phospholipids 12 g. glycerol anhydrous 22 g, water for injectionq.s. ad 1,000 mL. pH is adjusted with sodium hydroxide to pHapproximately 8. Intralipid® 20% contains in 1,000 mL: purified soybeanoil 200 g, purified egg phospholipids 12 g, glycerol anhydrous 22 g.water for injection q.s. ad 1,000 mL. pH is adjusted with sodiumhydroxide to pH approximately 8. Intralipid® 30% contains in 1,000 mL:purified soybean oil 300 g, purified egg phospholipids 12 g, glycerolanhydrous 16.7 g, water for injection q.s. ad 1,000 mL. pH is adjustedwith sodium hydroxide to pH approximately 7.5. These Intralipid®products are stored at controlled room temperature below 25° C. andshould not be frozen.

In one embodiment of the invention, the NDGA derivative is dissolved ordissolved and diluted in different carriers to form a liquid compositionfor oral administration into animals, including humans. For example, inone aspect of this embodiment, the NDGA derivative is dissolved in awater-soluble organic solvent such as a PEG 300, PEG 400 or a PEG 400monolaurate (the “PEG compounds”) or in PG. In another embodiment, theNDGA derivative is dissolved in a modified cyclodextrin, such as HP-β-CDor SBE-β-CD. In yet another embodiment, the present NDGA derivative issolubilized and/or diluted in a combination formulation containing a PEGcompound and HP-β-CD. In a further embodiment, the NDGA derivativeherein is dissolved in a modified cellulose such as HPMC, CMC or EC. Inyet another embodiment, the NDGA derivative herein is dissolved inanother combination formulation containing both a modified cyclodextrinand modified cellulose, such as, for example, HP-β-CD and HPMC orHP-β-CD and CMC.

In yet another embodiment, the NDGA derivative is dissolved in ionic,non-ionic or amphipathic surfactants such as Tween® 20, Tween® 80, TPGSor an esterified fatty acid. For example, the present compounds can bedissolved in TPGS alone, or Tween® 20 alone, or in combinations such asTPGS and PEG 400, or Tween® 20 and PEG 400.

In a further embodiment, the present NDGA derivative is dissolved in awater-insoluble lipid such as a wax, fat emulsion, for exampleIntralipid®, or oil. For example, the present compounds can be dissolvedin peppermint oil alone, or in combinations of peppermint oil withTween® 20 and PEG 400, or peppermint oil with PEG 400, or peppermint oilwith Tween® 20, or peppermint oil with sesame oil.

EC may be substituted or added in place of the HPMC or CMC in theforegoing examples; PEG 300 or PEG 400 monolaurate can be substituted oradded in place of PEG 400 in the foregoing examples; Tween® 80 may besubstituted or added in place of Tween® 20 in the foregoing examples;and other oils such as corn oil, olive oil, soybean oil, mineral oil orglycerol, may be substituted or added in place of the peppermint oil orsesame oil in the foregoing examples.

Further, heating may be applied, for example, heating to a temperatureof about 30° C. to about 90° C., in the course of formulating any ofthese compositions to achieve dissolution of the compounds herein or toobtain an evenly distributed suspension of the NDGA derivative.

In still a further embodiment, the NDGA derivative may be administeredorally as a solid, either without any accompanying carrier or with theuse of carriers. In one embodiment, the NDGA derivative is firstdissolved in a liquid carrier as in the foregoing examples, andsubsequently made into a solid composition for administration as an oralcomposition. For example, the NDGA derivative is dissolved in a modifiedcyclodextrin such as HP-β-CD, and the composition is lyophilized toyield a powder that is suitable for oral administration.

In a further embodiment, the NDGA derivative is dissolved or suspendedin a TPGS solution, with heating as appropriate to obtain an evenlydistributed solution or suspension. Upon cooling, the compositionbecomes creamy and is suitable for oral administration.

In yet another embodiment, the NDGA derivative is dissolved in oil andbeeswax is added to produce a waxy solid composition.

In general, in preparing the oral formulations, the NDGA derivativeherein is first solubilized before other excipients are added so as toproduce compositions of higher stability. Unstable formulations are notdesirable. Unstable liquid formulations frequently form crystallineprecipitates or biphasic solutions. Unstable solid formulationsfrequently appear grainy and clumpy and sometimes contain runny liquids.An optimal solid formulation appears smooth, homogenous, and has a smallmelting temperature range. In general, the proportions of excipients inthe formulation may influence stability. For example, too littlestiffening agent such as beeswax may leave the formulation too runny foran elegant oral formulation.

Hence, in general, for the liquid formulations of the present invention,the excipients used should be good solvents of the NDGA derivativeherein. In other words, the excipients should be able to dissolve theNDGA derivative without heating. The excipients should also becompatible with each other independent of the NDGA derivative such thatthey can form a stable solution, suspension or emulsion. Also, ingeneral, for the solid formulations of the present invention, theexcipients used should also be good solvents of the NDGA derivative toavoid clumps and non-uniform formulations. To avoid solid formulationsthat are too runny or heterogeneous in texture, which are undesirable,the excipients should be compatible with each other such that they forma smooth homogeneous solid, even in the absence of the NDGA derivative.

The present invention further relates to a method of producing thepharmaceutical composition of the present invention, the methodinvolving making or providing the NDGA derivative preferably in asubstantially purified form, combining the composition with apharmaceutically acceptable carrier or excipient, and formulating thecomposition in a manner that is compatible with the mode of desiredadministration.

The compounds and compositions of the present invention find use astherapeutic agents in situations, for example, where one wishes toprovide a treatment to a subject who has a proliferative disease such asa malignant, premalignant or benign tumor, a viral disease, aninflammatory disease, a metabolic disease or a vascular disease.

The compounds and compositions of the present invention can be used totreat a variety of tumors and cancers, including, without limitation,hematological malignancies such as leukemia, for instance acute orchronic lymphoblastic leukemia, acute or chronic myeloid leukemia, acuteor chronic lymphocytic leukemia, acute or chronic myelogenous leukemia,childhood acute leukemia, chronic lymphocytic leukemia, hairy cellleukemia, malignant cutaneous T-cells, mycosis fungoides, non-malignantfibrous cutaneous T-cell lymphoma, lymphomatoid papulosis, T-cell richcutaneous lymphoid hyperplasia, non-Hodgkin's lymphoma, Hodgkin'slymphoma, bullous pemphigoid, discoid lupus erythematosus, lichenplanus, adrenocortical carcinoma, anal cancer, bile duct cancer, bladdercancer, bone cancer, osteosarcoma/malignant fibrous histiocytoma,neurological tumors and malignancies such as neuroblastoma,glioblastoma, astrocytoma, gliomas, brain stem glioma, brain tumorependymoma, medulloblastoma, female and male breast cancer, carcinoidtumor gastrointestinal, carcinoma adrenocortical, carcinoma islet cell,clear cell cancer, clear cell sarcoma of tendon sheaths, colon cancer,colorectal cancer, cutaneous T-cell lymphoma, endometrial cancer,esophageal cancer. Ewing's family of tumors, extragonadal germ celltumor, extrahepatic bile duct cancer, eye cancer, intraocular melanoma,ductal cancer, eye cancer retinoblastoma, dysplastic oral mucosa,invasive oral tumor, gallbladder cancer, gastric (stomach) cancer,gastrointestinal carcinoid tumor, germ cell tumor extragonadal, germcell tumor, gestational trophoblastic tumor, hepatocellular (liver)cancer, hypopharyngeal cancer, intraocular melanoma, islet cellcarcinoma (endocrine pancreas), Kaposi's sarcoma, laryngeal cancer,liver cancer, lung tumors and cancers such as non-small cell lung cancerand small cell lung cancer, malignant mesothelioma, melanoma, merkelcell carcinoma, multiple endocrine neoplasia syndrome, mycosisfungoides, multiple mycloma, nasal cavity tumors, paranasal and sinuscancer, nasopharyngeal cancer, oral cavity and lip cancer, oropharyngealcancer, pancreatic cancer, parathyroid cancer, penile cancer,pheochromocytoma, pineal and supratentorial primitive neuroectodermal,tumors, pituitary tumor, pleuropulmonary blastoma, prostate cancer,rectal cancer, renal, pelvis and ureter transitional cell cancer,retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma softtissue adult, Sezary syndrome, skin cancer, small intestine cancer,testicular tumors and cancer, thymoma, thyroid cancer, urethral cancer,transitional and squamous cell urinary carcinoma, gynecological tumorsand cancer such as cervical cancer, ovarian tumors and cancer, ovarianepithelial cancer, ovarian germ cell tumor, uterine cancer, endometrialcancer, vaginal cancer, vulvar cancer, Waldenström's macroglobulinemia.Wilms' tumor, liver tumors including hepatocellular carcinoma (“HCC”)and tumors of the biliary duct, other lung tumors including small celland clear cell cancers, sarcomas in different organs; as well as othercancers and tumors.

Non limiting examples of viral diseases that may be treated effectivelyby the NDGA derivatives of the present invention, include for exampleand without limitation viral infections caused by human immunodeficiencyvirus (HIV), human papillomaviruses (HPV)(all subtypes), herpes simplexvirus 1 and 2 (HSV-1 and HSV-2), Varicella Zoster virus,cytomegalovirus. Epstein Barr virus, pox viruses (smallpox, cowpox,monkeypox, vaccinia), orthohepadnavirus, JC virus, and BK virus, amongothers.

Non-limiting examples of inflammatory diseases that may be treatedeffectively by the NDGA derivatives of the present invention include,for instance and without limitation, rheumatoid arthritis,osteoarthritis, psoriasis, sarcoidosis, systemic lupus erythematosis.Stills disease, cystic fibrosis, chronic obstructive pulmonary diseaseand inflammatory bowel diseases such as ulcerative colitis and Crohns,among others.

Non-limiting examples of metabolic diseases that may be treatedeffectively by the NDGA derivatives of the present invention include,for instance, diabetes mellitus (juvenile onset and adult onset),diabetes insipidis, syndrome X, hyperlipidemia, hypercholesterolemia,hypoglycemia, atheroma, ketoacidosis, Addisons disease, Cushingssyndrome, hyperparathyroidism, hyperthyroidism, leucodystrophy andporphyria, among others.

Non-limiting examples of vascular diseases that may be treatedeffectively by the NDGA derivatives of the present invention include,for instance and without limitation, arterial hypertension, pulmonaryarterial hypertension, cardiovascular disease and macular degeneration,among others.

As mentioned above, an effective amount of the NDGA derivative isadministered to the host, where “effective amount” means a dosagesufficient to produce a desired result. In some embodiments, the desiredresult is at least a reduction in one or more symptoms of the viralinfection or the inflammatory, metabolic, proliferative or vasculardisease. Typically, the compositions of the present invention willcontain from less than about 0.1% up to about 99% of the activeingredient, that is, the NDGA derivative of the present invention;optionally, the present invention will contain about 5% to about 90% ofthe active ingredient. The appropriate dose to be administered dependson the subject to be treated, such as the general health of the subject,the age of the subject, the state of the disease or condition, theweight of the subject, for example. Generally, about 0.1 mg to about 500mg may be administered to a child and about 0.1 mg to about 5 grams maybe administered to an adult. The NDGA derivative can be administered ina single or, more typically, multiple doses as frequently and over sucha time period as needed to treat the disease. Preferred dosages for agiven agent are readily determinable by those of skill in the art by avariety of means in view of the present disclosure. Other effectivedosages can be readily determined by one of ordinary skill in the art inview of the present disclosure through routine trials establishing doseresponse curves. The amount of NDGA derivative will, of course, varydepending upon the particular NDGA derivative used, as well as thenature of the formulation containing the NDGA derivative, and the routeof administration, the size and condition of the subject, the nature andextent of the disease, etc.

The frequency of administration of the NDGA derivative, as with thedoses, will be determined by the care giver based on age, weight,disease status, health status and patient responsiveness. Thus, theagents may be administered one or more times daily or as appropriate foras long as needed as conventionally determined.

Kits with multiple or unit doses of the NDGA derivative are included inthe present invention. In such kits, in addition to the containerscontaining the multiple or unit doses of the compositions containing theNDGA derivative will be instructions for its use for a given indicationsuch as an informational sheet or package insert with instructionsdescribing the use and attendant benefits of the drugs in treating thepathological condition of interest, such as any of a number ofinflammatory, metabolic, vascular or proliferative diseases or a viralinfection.

The present invention will now be described in greater detail withreference to the following specific, non-limiting working examples,except as noted where the examples are indicated as being prophetic.

General Procedure

All reactions were carried out in oven-dried glassware (120° C.) underan atmosphere of nitrogen, unless indicated otherwise. Acetone,dichloromethane, 1,4-dioxane, ethyl acetate, hexane, and tetrahydrofuranwere purchased from Mallinckrodt Chemical Co. Acetone was died with 4 Åmolecular sieves and distilled. Dichloromethane, ethyl acetate, andhexane were dried and distilled from CaH₂. 1,4-Dioxane andtetrahydrofuran were dried by distillation from sodium and benzophenoneunder an atmosphere of nitrogen. Nordihydroguaiaretic acid was purchasedfrom Fluka Chemical Co. 4-(2-Chloroethyl)morpholine hydrochloride,4-(3-chloropropyl)morpholine hydrochloride, 1-(3-chloropropyl)piperidinemonohydrochloride, 1-(2-chloroethyl)piperidine monohydrochloride,2-chloroethanol, (2-chloroethoxy)ethene, 1-(2-chloroethyl)pyrrolidinehydrochloride, N,N′-dicyclohexylcarbodiimide (DCC),4-dimethylaminopyridine (DMAP), and potassium carbonate were purchasedfrom Aldrich Chemical Co.

The melting point was obtained with a Büchi Labortechnik AG 535melting-point apparatus. Analytical thin layer chromatography (TLC) wasperformed on precoated plates (silica gel 60 F-254), purchased fromMerck Inc. Gas chromatographic analyses were performed on aHewlett-Packard 5890 Series II instrument equipped with a 25-mcrosslinked methyl silicone gum capillary column (0.32 mm i.d.).Nitrogen gas was used as a carrier gas and the flow rate was keptconstant at 14.0 mL/min. The retention time t_(R) was measured under thefollowing conditions: injector temperature 260° C., isothermal columntemperature 280° C. Gas chromatography and low resolution mass spectralanalyses were performed on a Agilent Technology 6890N Network GC Systemequipped with a Agilent 5973 Network Mass Selective Detector andcapillary HP-1 column. Purification by gravity column chromatography wascarried out by use of Merck Reagents Silica Gel 60 (particle size0.063-0.200 mm, 70-230 mesh ASTM). Purity of all compounds was >99.5%,as checked by HPLC or GC.

Ultraviolet (UV) spectra were measured on a Hitachi U3300 UV/VISspectrophotometer. Infrared (IR) spectra were measured on a JascoFT-IR-5300 Fourier transform infrared spectrometer. The wave numbersreported were referenced to the polystyrene 1601 cm⁻¹ absorption.Absorption intensities were recorded by the following abbreviations: s,strong; m, medium; w, weak. The fluorescent intensity was measured on aHitach F-4500 Florescence Spectmphotometer. Proton NMR spectra wereobtained on a Varian Mercury-400 (400 MHz) spectrometer by use ofchloroform-d as the solvent and sodium 3-(trimethylsilyl)propionate asinternal standard. Carbon-13 NMR spectra were obtained on a VarianMercury-400 (100 MHz) spectrometer by use of chloroform-d or D₂O as thesolvent. Carbon-13 chemical shifts were referenced to the center of theCDCl₃ triplet (δ 77.0 ppm). Multiplicities are recorded by the followingabbreviations: s, singlet; d, doublet; t, triplet; q, quartet; m,multiplet; J, coupling constant (hertz). High-resolution mass spectrawere obtained by means of a JEOL JMS-HX 110 mass spectrometer.Electrospray ionization mass spectrometry (ESI-MS) analyses wereperformed on a quadrupole ion trap mass analyzer fitted with anelectrospray ionization source of Finnigan LCQ, Finnigan MAT.

Computation was performed on a Silicon Graphics O2+ workstation.Software Chemoffice Ultra 10.0 was used to draw chemical structures andsynthetic schemes. The software PCModel 7.5 was used for energyminimized with the consistent valence force field (CVFF) until themaximum derivative was less than 1.0 kcal mol⁻¹ Å⁻¹.

Example 1 Synthesis of1,4-bis{3,4-bis[3-(piperidin-1-yl)propoxy]phenyl}-2,3-dimethyl-(2R,3S)-butanefree base (C₅₀H₈₂N₄O₄, FW=803.21) “Compound A”; andtetrakis-hydrochloride salt (C₅₀H₈₆N₄O₄Cl₄, FW=949.05) “Compound B” Step1: Synthesis of N-(3-chloropropyl)-piperidine

N-(3-Chloropropyl)-piperidine hydrochloride (97% purity from AldrichChemicals) (100 g) was dissolved in water (150 mL) and saturated aqueouspotassium carbonate (250 mL) was slowly added to it. Also, 10N sodiumhydroxide (25 mL) and diethyl ether (250 mL) were added and the mixturewas stirred for one hour. The layers were separated; the organic layerwas dried over anhydrous potassium carbonate and concentrated on a BüchiLabortechnik AG Rotavapor® evaporator to give the title compound (77.2g, 94.4%), which was used without purification for the next reactions.

Step 2: Synthesis of1,4-bis{3,4-bis[3-(piperidin-1-yl)propoxy]phenyl}-2,3-dimethyl-(2R,3S)-butane“Compound A”

To a solution of NDGA (15.0 g, 48.0 mmol) in anhydrous DMF (1 L) wasadded 60% suspension of sodium hydride in paraffin (10.4 g, 260 mmol,5.4 equiv.) and the mixture was heated at 65° C. for one hour. Then themixture was cooled to room temperature, compoundN-(3-chloropropyl)-piperidine (39.0 g, 241 mmols, 5.0 equiv.) and sodiumiodide (7.2 g, 48 mmol, 1 equiv.) were added and the mixture was allowedto stir at room temperature for 120 hours. TLC indicated completeconversion to the product.

The workup of the reaction was carried out by slow addition of thereaction mixture to water (3 L) and diethyl ether (2.5 L). The aqueouslayer was extracted again with diethyl ether (2 L). The combined organicextracts were washed with brine (750 mL), dried over anhydrous sodiumsulfate and concentrated under reduced pressure. The crude was purifiedby silica gel column chromatography. The column was built using silicagel (500 g) and solvent mixture ethyl acetate:methanol:triethylamine(93:2:5) and eluted with the gradient ethylacetate:methanol:triethylamine (93:2:5 to 91:4:5) to give the product aswhite solid (28.1 g, yield 76.6%). This product was crystallized fromethyl acetate:hexane mixture to give crystalline compound (22.5 g).

m.p. 84-86° C. HPLC purity—99.47%.

¹H NMR (CDCl₃, 300 MHz), δ=0.81 (d, J=6.6 Hz, 6H), 1.35-1.50 (m, 8H),1.53-1.65 (m, 16H), 1.67-1.80 (m, 2H), 1.90-2.2.03 (m, 8H), 2.40-2.60(m, 26H), 2.72 (m, 2H), 3.99 (t. J=6.5 Hz, 4H), 4.00 (t, J=6.5 Hz, 4H),6.65 (dd, J=1.9, 8.0 Hz, 2H), 6.67 (d, J=1.9 Hz, 2H), 6.79 (d, J=8.0 Hz,2H); consistent with the structure.

¹³C NMR (CDCl₃, 75 MHz): δ=16.1, 24.5, 26.0, 27.0, 38.9, 39.4, 54.7,56.2, 67.8, 68.0, 114.1, 115.2, 121.3, 134.9, 147.0, 148.8; consistentwith the structure.

Analysis: Calculated for C₅₀H₈₂N₄O₄C, 74.76; H, 10.29; N, 6.98. Found C,75.07; H, 10.59; N, 6.91.

Step 3: Synthesis of1,4-bis{3,4-bis[3-(piperidin-1-yl)propoxy]phenyl}-2,3-dimethyl-(2R,3S)-butanetetrakis-hydrochloride salt “Compound B”

To an ice cooled (0-5° C.) solution of aqueous concentrated HCl (7.2 mLof 11 N HCl, 79 mmol, 24 mole equiv.) in 95% ethanol (21 mL) was addeddrop-wise a solution of1,4-bis{3,4-bis[3-(piperidin-1-yl)propoxy]phenyl}-2,3-dimethyl-(2R,3S)-butane(2.658, 3.30 mmols) in 95% ethanol (21 mL). The solution was allowed tostir at 0-5° C. for three hours and the solvent was removed on a rotaryevaporator while keeping the temperature of the water bath at 45° C. Thehydrochloride salt was dried under high vacuum for 48 hours. The crudeproduct was crystallized from ethanol:ether to give 2.20 g of theproduct (70.3% yield) after drying under high vacuum for 72 hours. Theanalytical data for this product are given below.

m.p. 265-270° C. (dec.)

HPLC purity: 99.2% (% peak area); Moisture content by Karl Fishermethod: 2.3938%. Elemental Analysis—C₅₀H₈₆N₄O₄Cl₄, calculated: C, 63.27;H, 9.13; N, 5.90. Found: C, 63.60; H, 9.59; N, 5.73. Chlorine elementalanalysis by titration method (anhydrous basis): theory: 14.94%. found:14.97% (100.3% of the theory).

1H-NMR (D₂O, 300 MHz): δ=0.65-0.80 (ss, 6H), 1.40-2.0 (m, 26H),2.10-2.15 (m, 8H), 2.18-2.24 (m, 2H), 2.56-2.61 (m, 2H), 2.70-3.05 (m,8H), 3.05-3.20 (m, 8H), 3.20-3.60 (m, 8H), 4.05 (t, J=4.2 Hz, 8H), 6.71(dd, J=1.8, 8.4 Hz, 2H), 6.89 (d, J=1.8 Hz, 2H), 6.92 (d. J=8.4 Hz, 2H).

¹³C NMR (CDCl₃, 75 MHz) δ=16.0 (CH₃), 23.4 (CH₂), 24.5 (CH₂) 27.5 (CH₂),38.8 (Ar—CH₂), 39.4 (CH), 54.6 (NCH₂), 54.9 (NCH₂), 68.1 (OCH₂), 113.4(Ar), 114.1 (Ar), 121.4 (Ar), 135.0 (Ar), 146.8 (Ar), 148.5 (Ar) ppm;consistent with structure.

LC-MS, m/e=839 (M+K), 803 (M+) consistent with free base C₅₀H₈₂N₄O₄.

Example 2 Synthesis of1,4-bis{3,4-bis[4-(N-piperidino)butoxyl]phenyl}-2,3-dimethyl-(2R,3S)-butanefree base (C₅₄H₉₀O₄N₄, FW=859.32)—“Compound C”; andtetrakis-hydrochloride salt (C₅₄H₉₀O₄N₄.4Cl, FW=1005.16)—“Compound D”Step 1: Synthesis of N-(4-chlorobutyl)piperidine

In a 2 L three-necked round bottomed flask was added piperidine (85.15g, 1.0 mol) acetone (1000 mL), and anhydrous potassium carbonate (276.42g, 2.0 mol, 2.0 equiv.). The flask was equipped with a mechanicalstirrer and a condenser. 4-chloro-1-bromo-butane (188.6 g, 1.1 mol, 1.1equiv.) was added dropwise under continuous stirring at room temperatureover a period of 30 min. The suspension mixture was then stirred at 40°C. The progress of the reaction was monitored by TLC, which confirmedthe reaction was completed after 4 hours. The mixture was cooled to roomtemperature and the insoluble materials were removed by filtration andwashed with dichloromethane (2×100 mL). The combined filtrates wereconcentrated under vacuum until it became dry. The residue was thenmixed with dichloromethane (500 mL). Insoluble materials were removed byfiltration and washed by dichloromethane (2×100 mL). The combinedfiltrate and washings were concentrated and purified through a flashsilica gel column, which gave the expected product as light yellow oil(98.39 g, 56% yield).

¹H NMR (CDCl₃, 300 MHz), δ=1.37 (m, 2H. CH₂), 1.60 (m, 8H, 4 CH₂), 2.46(t, J=6.5 Hz, 4H, 2CH₂N), 2.89 (t, J=6.8 Hz, 2H, CH₂N), 3.56 (t, J=7.2Hz, CH₂Cl) ppm; consistent with the structure.

¹³C NMR (CDCl₃, 75 MHz), δ=24.5, 25.1, 25.5, 26.1, 44.1 (CH₂NCl), 53.1(CH₂N), 55.4 (CH₂N) ppm; consistent with the structure.

MS (EI), m/c=176 (M+1); consistent with the structure.

Step 2: Synthesis of1,4-bis{3,4-bis[4-(N-piperidino)butoxyl]phenyl}-2,3-dimethyl-(2R,3S)-butane“Compound C”

To a solution containing nordihydroguaiaretic acid (NDGA, 602 mg, 2.0mmol) and lithium hydroxide monohydrate (1.0 g, 24.0 mmol, 12 equiv.) intert-butanol (100 mL) was added N-(4-chlorobutyl)piperidine (2.11 g,12.0 mmol, 6.0 equiv.). The solution was heated at 50° C. undercontinuous stirring. The reaction was monitored byTLC(CH₂Cl₂:MeOH:Et₃N=95(3:2, V/V/V), which confirmed the reaction wascompleted after 20 hours. The reaction suspension was cooled to roomtemperature, and partitioned with dichloromethane (200 mL) and water(100 mL). The mixture was shaken to mix well. The organic phase wasseparated and the aqueous phase was extracted with dichloromethane(2×100 mL). The organic layer and extracts were combined and washed withaqueous saturated sodium bicarbonate (100 mL), and brine (10 mL). Afterdrying over anhydrous sodium sulfate, the solution was concentratedunder reduced vacuum. The residue was purified through a silica gelcolumn using dichloromethane, methanol and triethylamine (95:3:2, V/V/V)as an eluent, which gave the expected product as a white semi-solidmaterial (704.6 mg, yield 41%).

HPLC purity—98.5%.

¹H NMR (CDCl₃, 300 MHz), δ=0.81 (d, J=6.6 Hz, 6H, 2 CH₃), 1.35-1.50 (m,8H, 4CH₂), 1.53-1.65 (m, 24H, 12 CH₂), 1.67-1.80 (m, 2H), 1.90-2.2.03(m, 8H), 2.40-2.60 (m, 26H), 2.72 (m, 2H), 3.99 (t, J=6.5 Hz, 4H), 4.00(t, J=6.5 Hz, 4H), 6.65 (dd, J=1.9, 8.0 Hz, 2H), 6.67 (d, J=1.9 Hz, 2H),6.79 (d, J=8.0 Hz, 2H) ppm; consistent with the structure.

¹³C NMR (CDCl₃) 16.1, 24.5, 25.4, 26.0, 27.0, 38.9, 39.4, 54.7, 56.2,67.8, 68.0, 114.1, 115.2, 121.3, 134.9, 147.0, 148.8 ppm; consistentwith the structure.

Analysis: Calculated for C₅₄H₉₀N₄O₄ (859.32) C, 75.48; H, 10.56; N,6.52. Found C, 75.07; H, 10.59; N, 6.91; consistent with the structure.

Step 3: Synthesis of1,4-bis{3,4-bis[4-(N-piperidino)butoxyl]phenyl}-2,3-dimethyl-(2R,3S)-butanetetrakis-hydrochloride salt “Compound D”

To an ice cooled (0-5° C.) solution of aqueous concentrated HCl (2.2 mLof 11 N HCl, 24 mmol, 24 mole equiv.) in 95% ethanol (7 mL) was addeddrop-wise a solution of1,4-bis(3,4-bis[4-(piperidin-1-yl)butoxy]phenyl-2,3-dimethyl-(2R,3S)-butane(859.32 mg, 1.0 mmols) in 95% ethanol (7 mL). The solution was allowedto stir at 0-5° C. for three hours and the solvent was removed on rotaryevaporator while keeping the temperature of the water bath at 45° C. Thehydrochloride salt was dried under high vacuum for 48 hours. The crudeproduct was then crystallized from ethanol:ether to give 736.9 mg of theproduct (73.3% yield) after drying under high vacuum for 72 hours. Theanalytical data for this product are given below.

m.p. 225-230° C. (dec.).

HPLC purity: 99.2% (% peak area). Elemental Analysis—C₅₄H₉₀O₄N₄4 HCl,FW=1005.16, calculated: C, 64.53; H, 9.43; N, 5.57. Found: C, 64.23; H,9.21; N, 5.43. Chlorine elemental analysis by titration method(anhydrous basis): theory: 14.11%. found: 14.21% (100.7% of the theory).

¹H-NMR (D₂O, 400 MHz): δ=0.76 (d, J=6.4 Hz, 6H, 2×CH₃), 1.28-1.37 (m,8H), 1.36-1.46 (m, 8H, 4× piperidine CH₂), 1.43-2.56 (m, 16H, 8×piperidine CH₂), 1.55-1.68 (m, 2H, 2×CH), 2.36-2.51 (m, 16H, 8×piperidine CH₂N), 2.64 (dd, J=13.2, 1.2 Hz, 2 E, 2×ArCH), 2.71 (t, J=6.0Hz, 8H, 4×CH₂N), 4.03 (t, 8H, 4×CH₂O), 6.60-6.75 (m, 6H, 6×AlH) ppm;consistent with structure.

¹³C NMR (D₂O, 100 MHz), δ=15.93, 23.85, 24.00, 25.06, 25.72, 38.92,39.31, 54.87, 54.97, 57.87, 67.13, 67.28, 113.90, 114.96, 121.38,134.71, 146.75, 148.48 ppm; consistent with structure.

MS (EI), m/e=859 (M+), consistent with free base C₅₄H₉₀N₄O₄.

Example 3 Synthesis of1,4-bis{3,4-bis(2-methyl-thiazol-4-yl-methoxy)phenyl}-2,3-dimethyl-(2R,3S)-butanefree base (C₃₈H₄₆N₄O₄S₄, FW=747.02) “Compound E”; tetrakis-hydrochloridesalt (C₃₈H₄₆N₄O₄S₄.4HCl, FW=892.87) “Compound F” Step 1: Synthesis of1,4-bis{3,4-bis(2-methyl-thiazol-4-yl-methoxy)phenyl}-2,3-dimethyl-(2R,3S)-butane“Compound E”

Preparation of 4-chloromethyl-2-methylthiazole from its hydrochloride:4-chloromethyl-2-methyl thiazole hydrochloride (25 g, 135 mmol) wasadded 50% aqueous potassium carbonate (100 mL) and ether (400 mL). Themixture was stirred at room temperature for 15 min. The organic layerwas separated, dried over anhydrous potassium carbonate, andconcentrated to dryness under reduced vacuum (30-40 mm Hg) at 25° C. Theresidue was dried under high vacuum (0.1-0.5 mm Hg) overnight at roomtemperature to give 4-chloromethyl-2-methylthiazole (20 g, 99.5% yield).

Preparation of1,4-bis{3,4-bis(2-methyl-thiazol-4-yl-methoxy)phenyl}-2,3-dimethyl-(2R,3S)-butane:To an ice-cooled solution of NDGA (1.50 g, 4.96 mol) in DMF (50 mL) wasadded 60% suspension of sodium hydride in paraffin (1.98 g=1.19 g NaH,49.6 mmol, 10 mole equiv.). The mixture was stirred at 0° C. for 30 min.and at room temperature for 30 min. Then a solution of4-chloromethyl-2-methylthiazole (5.86 g, 39.69 mmol, 8.0 mole equiv.) inDMF (20 mL) was added and the reaction mixture was allowed to stir for16 h at room temperature. The reaction mixture was added to thesaturated aqueous ammonium chloride (300 mL) and ether (600 mL). Aftershaking, the organic layer was separated, and the aqueous layer wasextracted with ether (2×200 mL). The combined organic layer and extractswere washed with water (100 mL), brine (100 mL), and dried overanhydrous sodium sulfate and concentrated to dryness under reducedvacuum (30-40 mm Hg) at 25° C. The residue was dissolved in a minimumamount of dichloromethane and purified by silica gel flashchromatographic column using hexane:ethyl acetate (50:50 to 0:100) aseluant to give expected product as white solid (1.56 g, 42.09% yield).It was further purified by crystallization from acetate-hexane.

Mp 85-87° C., HPLC purity: 98.9%.

¹H NMR (300 MHz, CDCl₃): δ=0.84 (d, J=6.4 Hz, 6H, 2×CH₃), 2.01 (m, 2H,2×CH), 2.65-2.70 (m, 4H, 2×Ar—CH₂) 2.81 (s, 6H, 2×CH₃), 5.23 (d, J=20.7Hz, 8H, 4×OCH₂), 6.70-6.84 (m, 6H, 6×Ar—H), 7.07 (s, 4H, 4-Ar—H) ppm;consistent with structure.

¹³C NMR (75 MHz, CDCl₃): δ=16.5 (CH₃), 19.5 (CH₃), 38.6 (Ar—CH₂), 39.4(CH), 72.6 (OCH₂), 112.1 (Ar), 113.8 (Ar), 115.4 (Ar), 122.8 (Ar), 131.1(Ar), 135.0 (Ar), 147.4 (Ar), 149.5. (Ar), 159.8 (Ar), 165.3 (Ar) ppm;consistent with structure.

LC-MS, m/e=770 (M+Na⁺), 747 (M+).

Analysis: calculated for C₃₈H₄₂N₄O₄S₄, FW=747.02; C, 61.10; H, 5.67; N,7.50. Found C, 60.86; H, 5.86; N, 7.31.

Step 2: Synthesis of1,4-bis{3,4-bis(2-methyl-thiazol-4-yl-methoxy)phenyl}-2,3-dimethyl-(2R,3S)-butanetetrakis-hydrochloride salt “Compound F”

To a solution of1,4-bis{3,4-bis(2-methyl-thiazol-4-yl-methoxy)phenyl}-2,3-dimethyl-(2R,3S)-butane(0.50 g, 0.6 mmol) in acetone (20 mL), hydrochloride gas was slowlybubbled through the solution at room temperature for 10 min. undercontinuous stirring. The precipitates were collected by suctionfiltration. The collected materials were dissolved in a minimum amountof methanol and precipitated by anhydrous ether. The solid material wasthen collected by suction filtration, and dried under vacuum overnightto give 0.51 g (85% yield) of the expected tetrakis-hydrochloride saltas a white solid.

m.p. 91-92° C. HPLC purity: 98.9% (peak area purity)

¹H NMR (300 MHz, methanol-d₄): δ=0.74 (d, J=6.4 Hz, 6H, 2×CH₃), 2.11 (m,2H, 2×CH), 2.55-2.60 (m, 4H, 2×Ar—CH₂) 2.75 (s, 6H, 2×CH₃), 5.28 (d,J=20.7 Hz, 8H, 4×OCH₂), 6.77-6.87 (m, 6H, 6×Ar—H), 7.09 (s, 4H, 4-Ar—H),10.56 (brs, NH) ppm; consistent with structure.

¹³C NMR (75 MHz, methanol-d₄): δ=16.7 (CH₃), 19.5 (CH₃), 38.6 (Ar—CH₂),39.4 (CH), 72.6 (OCH₂), 112.1 (Ar), 113.8 (Ar), 115.4 (Ar), 122.8 (Ar),131.1 (Ar), 135.0 (Ar), 147.4 (Ar), 149.5 (Ar), 159.8 (Ar), 165.3 (Ar)ppm; consistent with structure.

LC-MS, m/e=770 (M+Na⁺), 747 (M+) consistent with structure of the parentcompound.

Analysis: calculated for C₃₈H₄₂N₄O₄S₄.4HCl, FW=892.87; C, 51.12; H,5.19; N, 6.27. Found C, 50.86; H, 5.06; N, 6.01.

Example 4 Synthesis of1,4-bis{3,4-bis(2-(N,N′-dimethylamino)-ethoxy)phenyl}-2,3-dimethyl-(2R,3S)-butanefree base (C₃₄H₅₈N₄O₄, FW=586.85) “Compound G”; tetrakis-hydrochloridesalt (C₃₄H₅₈N₄O₄.4HCl, FW=732.69) “Compound H” Step 1: Synthesis of1,4-bis{3,4-bis(2-(N,N′-dimethylamino)-ethoxy)phenyl}-2,3-dimethyl-(2R,3S)-butane“Compound G”

To a solution of NGDA (1.50 g, 4.96 mmol) in acetone (150 mL) were addedanhydrous potassium carbonate (6.84 g, 49.6 mmol, 10 mole equiv.), andN,N-dimethyl-N-(2-chloroethyl)amine hydrochloride (4.28 g, 29.7 mmol, 6mole equiv.). The mixture was stirred under reflux for 12 hours. Theprogress of the reaction was monitored by TLC(dichloromethane:methanol=95:5, v:v), and showed a considerable amountof NDGA unreacted. Thus, additional anhydrous potassium carbonate (6.84g, 49.6 mmol, 10 mole equiv.), and N,N-dimethyl-N-(2-chloroethyl)aminehydrochloride (4.28 g, 29.7 mmol, 6 mole equiv.) were added and themixture was stirred at reflux for another 64 hours. TLC monitoringshowed the reaction was complete. The mixture was cooled to roomtemperature. The insoluble materials were removed by suction filtration,and washed with acetone (2×50 mL). The filtrate and washings werecombined and concentrated to dryness under reduced vacuum (30-40 mm Hg)at 25° C. The residue was dissolved in a minimum amount ofdichloromethane. The insoluble materials were removed by suctionfiltration. The filtrate was purified by silica gel flashchromatographic column using gradient elutiondichloromethane:methanol:triethylamine (from 94:1:5 to 85:10:5) to givethe expected product as white solid (1.56 g, 53.6% yield). It wasfurther purified by crystallization from acetate-hexane.

Mp 98-100° C., HPLC purity: 99.1% (peak area purity).

¹H NMR (300 MHz, CDCl₃): δ=0.81 (d, J=6.4 Hz, 6H, 2CH₃), 1.95 (m, 2H,2×CH), 2.60-2.65 (m, 4H, 2 Ar—CH₂), 2.72 (t, J=6.1 Hz, 8H, 4×NCH₂) 2.86(s, 6H, 2×NCH₃), 4.08 (t, J=6.1 Hz, 8H, 4×OCH₂), 6.60-6.75 (m, 6H,6×Ar—H) ppm; consistent with structure.

¹³C NMR (75 MHz, CDCl₃): δ=15.2 (CH₃), 36.6 (Ar—CH₂), 38.7 (CH), 45.5(NCH₃), 56.7 (NCH₃), 65.4 (OCH₂), 112.8 (Ar), 114.4 (Ar), 121.8 (Ar),132.8 (Ar), 146.4 (Ar), 147.5 (Ar) ppm; consistent with structure.

LC-MS, m/e=609 (M+Na⁺), 586 (M+); consistent with structure.

Analysis: calculated for C₃₄H₅₈N₄O₄, FW=586.85; C, 69.59; H, 9.96; N,9.55. Found C, 69.86; H, 9.76; N, 9.31.

Step 2: Synthesis of1,4-bis{3,4-bis(2-(N,N′-dimethylamino)-ethoxy)phenyl}-2,3-dimethyl-(2R,3S)-butanetetrakishydrochloride salt “Compound H”

To a solution of1,4-bis(3,4-bis(2-(N,N′-dimethylamino)-ethoxy)phenyl)-2,3-dimethyl-(2R,3S)-butane(0.50 g, 0.85 mmol) in acetone (20 mL), hydrochloride gas was slowlybubbled through the solution at room temperature for 10 min. undercontinuous stirring. The precipitates were collected by suctionfiltration. The collected materials were dissolved in a minimum amountof methanol and precipitated by anhydrous ether. The solid material wasthen collected by suction filtration, and dried under vacuum overnightto give 0.44 g (70% yield) of the expected tetrakis-hydrochloride saltas a white solid.

m.p. 78-80° C. HPLC purity: 99.2% (peak area purity).

¹H NMR (300 MHz, methanol-d₄): δ=0.76 (d, J=6.4 Hz, 6H, 2×CH₃), 1.85 (m,2H, 2×CH), 2.63-2.69 (m, 4H, 2×Ar—CH₂), 2.75 (t, J=6.1 Hz, 8H, 4′NCH₂)2.96 (s, 6H, 2×NCH₁), 4.01 (t, J=6.1 Hz, 8H, 4×OCH₂), 6.65-6.77 (m, 6H,6×Ar—H), 10.55 (br s, NH) ppm; consistent with structure.

¹³C NMR (75 MHz, methanol-d₄): δ=16.2 (CH₃), 35.6 (Ar—CH₂), 37.9 (CH),46.6 (NCH₃), 55.9 (NCH₃), 64.7 (OCH₂), 112.5 (Ar), 115.4 (Ar), 122.8(Ar), 131.7 (Ar), 145.4 (Ar), 146.5 (Ar) ppm; consistent with structure.

LC-MS, m/e=609 (M+Na⁺), 586 (M+) consistent with structure. Analysis:calculated for C₃₄H₅₈N₄O₄.4HCl, FW=732.69. C, 55.73; H, 8.53; N, 7.65.Found C, 55.38; H, 8.36; N, 7.31.

Example 5 Synthesis of1,4-bis{3,4-bis(2-hydroxyethoxy)phenyl}-2,3-dimethyl-(2R,3S)-butane freebase (C₂₆H₃₈O₈, FW=478.58) “Compound I”

Method 1: (for Similar Synthetic Procedures See: J. Chem. Soc. Chem.Commun. 1987, (3), 223-224; J. Am. Chem. Soc. 1981, 103, 2361)

To a solution of NDGA (2.0 g, 6.61 mmol) in anhydrous dimethyl formamide(DMF) (20 mL) were added ethylene carbonate (3.49 g, 39.69 mmol, 6.0mole quiv.) and tetraethylammonium bromide (69 mg, 0.33 mmol, 0.05 moleequiv). The mixture was stirred at 140° C. The reaction was monitored byTLC (dicholoromethane:methanol=9:1, V/V), which showed the reactioncompleted after 36 hours. DMF was then removed under reduced vacuum(20-30 mm Hg) at 80-90° C. to dryness. The residue was then taken up bydichloromethane (200 mL), and washed by aqueous sodium bicarbonate (50mL), and brine (2×50 mL). After drying over anhydrous sodium sulfate,the solvent was removed under reduced vacuum (30-40 mm Hg) at 30-40° C.to dryness. The residue was dissolved in a minimum amount ofdichloromethane and purified by silica gel flash chromatographic columnusing gradient elution of dichloromethane:methanol (from 10:0 to 9:1,V/V) to give the expected product, which was further purified byre-crystallization from dichloromethane-ether to give pure product 0.88g (27.8% yield).

m.p. 120-122° C. with HPLC purity 99.4%.

¹H NMR (300 MHz, CDCl₃): δ=0.73 (d, J=6.4 Hz, 6H, 2×CH₃), 1.85 (m, 2H,2×CH), 2.35, 2.65 (mm, 4H, 2×Ar—CH₂), 3.74 (t, J=6.1 Hz, 8H, 4×OCH₂),3.93 (t, J=6.1 Hz, 8H, 4×OCH₂), 6.56-6.72 (m, 6H, 6×Ar—H) ppm;consistent with structure.

¹³C NMR (75 MHz, CDCl₃): δ=15.4 (CH₃), 35.2 (Ar—CH₂), 37.9 (CH), 61.8(OCH₂), 68.7 (OCH₂), 112.3 (Ar), 115.4 (Ar), 122.9 (Ar), 132.8 (Ar),146.4 (Ar), 148.1 (Ar) ppm; consistent with structure.

LC-MS, m/e=501 (M+Na⁺), 478 (M+); consistent with structure.

Analysis: calculated for C₂₆H₃₈O₈, FW=478.58; C, 65.25; H, 8.00. FoundC, 65.01; H, 8.34.

Method 2: (for Similar Synthetic Procedures See Synth. Commun. 2002,32(12), 1909-1915)

To a solution of NDGA (1.5 g, 4.96 mmol) in n-butanol (150 mL) wereadded a solution of sodium hydroxide (4.0 g, 99.20 mmol, 20 mole equiv.)in water (20 mL) and 2-chloroethanol (8.0 g, 99.22 mmol, 20 moleequiv.). The mixture was stirred under reflux. The reaction wasmonitored by TLC (dichlormethanol:methanol, 9:1, V/V), and showed to becompleted after 36 hours. The mixture was cooled to 0° C. and carefullyneutralized to pH 7.0 by conc. hydrochloric acid. The mixture wasconcentrated under reduced vacuum (30-40 mm Hg) at 70-80° C. to dryness.The residue was mixed and stirred with ethyl acetate (200 mL). Insolublematerials were removed by filtration and washing with ethyl acetate(2×50 mL). The filtrate and washings were combined, washed with brine(2×100 mL). After drying over anhydrous sodium sulfate, the mixture wasconcentrated under reduced vacuum (30-40 mm Hg) at 30-40° C. to dr mess.The residue was then dissolved in a minimum amount of dichloromethaneand purified by a silica gel flash chromatographic column using gradientelution dichloromethane:methanol (from 95:5 to 80:20, V/V) to give theexpected compound as white solid, which was further purified byre-crystallization from dichloromethane-ether to give the pure product1.32 g (55.6%), mp. 120-122° C. with HPLC purity 98.5%. Analytical datawere identical with the sample from Method 1.

Method 3: (for Similar Experimental Procedure See Bull. Korean Chem.Soc. 2004, 25(12), 1941-1944).

To a solution of NDGA (1.5 g, 4.96 mmol) in DMF (150 mL) were addedanhydrous potassium carbonate (13.70 g, 99.20 mmol, 20 mole equiv.) and(2-chloroethoxy)ethane (10.57 g, 99.22 mmol, 20 mole equiv.). Themixture was stirred at 130° C. The reaction was monitored by TLC(dichlormethanol:methanol, 9:1, V/V), and showed to be completed after48 hours. The mixture was concentrated under reduced vacuum (5-10 mm Hg)at 50-60° C. to dryness. The residue was mixed and stirred with ethylacetate (200 mL). Insoluble materials were removed by filtration andwashing with ethyl acetate (2×50 mL). The filtrate and washings werecombined, washed with brine (2×100 mL). After drying over anhydroussodium sulfate, the mixture was concentrated under reduced vacuum (30-40mm Hg) at 30-40° C. to dryness. The residue was then dissolved in aminimum amount of dichloromethane and purified by a silica gel flashchromatographic column using gradient elution dichloromethane:methanol(from 95:5 to 80:20, V/V) to give the expected compound as white solid,which was further purified by re-crystallization fromdichloromethane-ether to give the pure product 1.50 g (63.1% yield), mp.120-122° C. with HPLC purity 98.5%. Analytical data were identical withthe sample from Method 1.

Example 6 Synthesis of1,4-bis{3,4-bis[2-(2-hydroxyethoxy)ethoxyl]phenyl}-2,3-dimethyl-(2R,3S)-butanefree base (C₃₄H₅₄N₄O₁₂, FW=654.79) “Compound J”

To a solution of NDGA (1.5 g, 4.96 mmol) in DMF (150 mL) were addedanhydrous potassium carbonate (13.70 g, 99.20 mmol. 20 mole equiv.) and(2-chloroethoxy)ethanol (12.36 g, 99.22 mmol, 20 mole equiv.). Themixture was stirred at 130° C. The reaction was monitored by TLC(dichlormethanol:methanol, 9:1, V/V), and showed to be completed after72 hours. The mixture was concentrated under reduced vacuum (5-10 mm Hg)at 50-60° C. to dryness. The residue was mixed and stirred with ethylacetate (200 mL). Insoluble materials were removed by filtration andwashing with ethyl acetate (2×50 mL). The filtrate and washings werecombined, washed with brine (2×100 mL). After drying over anhydroussodium sulfate, the mixture was concentrated under reduced vacuum (30-40mm Hg) at 30-40° C. to dryness. The residue was then dissolved in aminimum amount of dichloromethane and purified by a silica gel flashchromatographic column using gradient elution dichloromethane:methanol(from 95:5 to 80:20, V/V) to give the expected compound, which wasfurther purified by re-crystallization from dichloromethane-ether togive the pure product 1.25 g (38.5% yield) as a light-yellow semi-solid,with HPLC purity 98.5%.

¹H NMR (300 MHz, CDCl₃): δ=0.74 (d, J=6.4 Hz, 6H, 2×CH₃), 1.88 (m, 2H,2×CH), 2.38, 2.68 (m, 4H, 2×Ar—CH₂), 3.60-4.01 (m, 32H, 16×OCH₂),6.58-6.74 (m, 6H, 6×Ar—H) ppm; consistent with structure.

¹³C NMR (75 MHz, CDCl₃): δ=15.4 (CH₃), 36.3 (Ar—CH₂), 38.5 (CH), 61.3(OCH₂), 69.7 (OCH₂), 70.7 (OCH₂), 71.2 (OCH₂), 112.4 (Ar), 114.1 (Ar),123.4 (Ar), 131.6 (Ar), 145.9 (Ar), 149.5 (Ar) ppm; consistent withstructure.

LC-MS, m/c=677 (M+Na⁺), 672 (M+H₂O), 655 (M+); consistent withstructure.

Analysis: calculated for C₃₄H₅₄O₁₂, FW=654.79; C, 62.37; H, 8.31. FoundC, 62.01; H, 8.14.

Example 7 Synthesis of Synthesis of1,4-bis[3,4-bis(2-fluoro-ethoxyl)phenyl]-2,3-dimethyl-(2R,3S)-butane(C₂₅H₃₄O₄F₄, FW=486.54) “Compound K”

To a suspension of Compound I (479 mg, 1.0 mmol) in dichloromethane (10mL) cooled in an ice water bath was added diethylaminosulfur trifluoride(DAST, 805 mg, 5 mmol). After the resulting yellowish solution wasstirred at room temperature for 10 hours, it was quenched with saturatedsodium bicarbonate (2 mL) under cooling. The organic layer was washedwith saturated brine, dried over MgSO₄(s), filtered, and concentratedunder reduced pressure. The residue was purified by flash columnchromatography on silica gel (10% methanol in dichloromethane as eluent)and the desired fraction was concentrated to give the expected compoundas an off white solid (277 mg, 0.47 mmol) in 57% yield.

m.p. 98-102° C., HPLC purity: 97.5% (peak area purity).

¹H NMR (CDCl₃, 400 MHz): δ=0.76 (d. J=6.4 Hz, 6H, 2×CH₃), 1.63-1.70 (m,2H, 2×CH), 2.18 (dd, J=13 Hz, 2H, 2×ArCH), 2.76 (dd, J=13.2, 4.6 Hz, 2H,2×ArCH), 4.09 (t, J=5.6 Hz, 8H, 4′CH₂O), 4.66 (m, J=47 Hz, 8H, 4×CH₂F),6.55-6.72 (m, 6H, 6×ArH); consistent with expected structure.

¹³C NMR (CDCl₃, 100 MHz): δ=15.90, 38.68, 39.23, 64.23, 64.50, 66.49,66.86, 113.75, 114.80, 121.56, 134.96, 146.63, 148.38; consistent withexpected structure.

MS (EI) m/e: 486 (M+), 496 (M+), 525 (M+K); consistent with expectedstructure.

Analysis: calculated for C₂H₃₄O₄F₄, FW=486.54; C, 64.18; H, 7.04. Found,C, 63.95; H, 6.85.

Example 8 Synthesis of1,4-bis{3,4-bis[4-(N-morpholino)butoxyl]phenyl}-2,3-dimethyl-(2R,3S)-butanefree base (C₅₀H₈₂N₄O₈, FW=867.21) “Compound L”; tetrakis-hydrochloridesalt (C₅₄H₈₂N₄O₈.4HCl, FW=1013.05) “Compound M” Step 1: Synthesis ofN-(4-chlorobutyl)morphine

In a 2 L three-necked round bottomed flask was added morpholine (87.12g, 1.0 mol) acetone (1000 mL), and anhydrous potassium carbonate (276.42g, g, 2.0 mol. 2.0 equiv.). The flask was equipped with a mechanicalstirrer and a condenser. 4-chloro-1-bromo-butane (188.6 g, 1.1 mol, 1.1equiv.) was added dropwise under continuous stirring at room temperatureover a period of 30 min. The suspension mixture was then stirred at 40°C. The progress of the reaction was monitored by TLC, which confirmedthe reaction was completed after 4 hours. The mixture was cooled to roomtemperature and the insoluble materials were removed by filtration andwashed with dichloromethane (2×100 mL). The combined filtrates wereconcentrated under vacuum until they became dry. The residue was thenmixed with dichloromethane (500 mL). Insoluble materials were removed byfiltration and washed by dichloromethane (2×100 mL). The combinedfiltrate and washings were concentrated and purified through a flashsilica gel column, which gave the expected product as a light yellow oil(80.83 g, 45.5% yield).

¹H NMR (CDCl₃, 300 MHz), 8=1.35 (m, 2H, CH₂), 1.75 (m, 2H, CH₂), 2.41(t, J=6.5 Hz, 4H, 2CH₂N), 2.97 (t, J=6.8 Hz, 2H, CH₂N), 3.65 (m, 6H, 2OCH₂, CH₂Cl) ppm; consistent with the structure.

¹³C NMR (CDCl₃, 75 MHz), δ=25.4, 25.8, 45.4, 53.2, 60.2, 65.5 ppm;consistent with the structure.

MS (EI), m/e=178 (M+1); consistent with the structure.

Step 2: Synthesis of1,4-bis{3,4-bis[4-(N-morphino)butoxyl]phenyl}-2,3-dimethyl-(2R,3S)-butane“Compound L”

To a solution containing NDGA (602 mg, 2.0 mmol) and lithium hydroxidemonohydrate (1.0 g, 24.0 mmol. 12 equiv.) in tert-butanol (100 mL) wasadded N-(4-chlorobutyl)morpholine: (2.13 g, 12.0 mmol, 6.0 equiv.). Thenthe solution was heated at 50° C. under continuous stirring. Thereaction was monitored by TLC (CH₂Cl₂:MeOH:Et₃N=92:6:2, V/V/V), whichconfirmed the reaction was completed after 20 hours. The reactionsuspension was cooled to room temperature, and partitioned withdichloromethane (200 mL) and water (100 mL). The mixture was shaken tomix well. The organic phase was separated and the aqueous phase wasextracted with dichloromethane (2×100 mL). The organic layer andextracts were combined and washed with aqueous saturated sodiumbicarbonate (100 mL), and brine (10 mL). After drying over anhydroussodium sulfate, the solution was concentrated under reduced vacuum. Theresidue was purified through a silica gel column using dichloromethane,methanol and triethylamine (92:6:2, V/V/V) as an eluent, which gave theexpected product as white semi-solid material (667.75 mg, 38.5% yield).

HPLC purity—99.2%. Elemental Analysis—C₅₀H₈₂N₄O₈, FW=867.21, calculated:C, 69.25; H, 9.53; N, 6.46. Found: C, 69.05; H, 9.21; N, 6.43;consistent with structure.

¹H NMR (CDCl₃, 400 MHz): δ=0.74 (d, J=6.4 Hz, 6H, 2×CH₃), 1.32-1.46 (m,16H) 1.62-1.71 (m, 2H, 2×CH), 2.26 (dd, J=13.4, 9.2 Hz, 2H, 2×ArCH),2.75 (dd, J=13.2, 4.6 Hz, 2H, 2×ArCH), 2.51-2.79 (m, 16H, 8× morpholineCH₂), 2.89 (t, J=12.2 Hz, 8H, 4×CH₂N), 3.56-3.76 (m, 16H, 8× morpholineCH₂), 4.11 (t, J=5.6 Hz, 8H, 4×CH₂O), 6.47-6.69 (m, 6H, 6×ArH) ppm,consistent with structure.

¹³C NMR (CDCl₃, 100 MHz): δ=16.96, 24.31, 24.91, 38.77, 40.18, 54.31,55.79, 56.46, 60.59, 65.60, 67.58, 67.70, 115.68, 116.09, 122.39,135.75, 145.39, 149.29 ppm; consistent with structure.

MS (FAB) m/e: 867 (M+), 434 (½M+), consistent with the structure(C₅₄H₈₂O₈N₄).

Analysis: Calculated for C₅₀H₈₂N₄O₈ (867.21) C, 69.25; H, 9.53; N, 6.46.Found C, 68.94; H, 9.39: N, 6.91, consistent with the structure.

Step 3: Synthesis of1,4-bis{3,4-bis[4-(N-morphino)butoxyl]phenyl}-2,3-dimethyl-(2R,3S)-butanetetrakis-hydrochloride “Compound M”

To an ice cooled (0-5° C.) solution of aqueous concentrated HCl (2.2 mLof 11 N HCl, 24 mmol, 24 mole equiv.) in 95% ethanol (7 mL) was addeddropwise a solution of1,4-bis(3,4-bis[4-(N-morpholino)butoxy]phenyl)-2,3-dimethyl-(2R,3S)-butane(867.21 mg, 1.0 mmols) in 95% ethanol (7 mL). The solution was allowedto stir at 0-5° C. for three hours and the solvent was removed on arotary evaporator while keeping the temperature of the water bath at 45°C. The hydrochloride salt was dried under high vacuum for 48 hours. Thecrude product was then crystallized from ethanol:ether to give 691.9 mgof the product (68.3% yield) after drying under high vacuum for 72hours. The analytical data for this product are given below.

m.p. 215-220° C. (dec.).

HPLC purity: 98.2% (% peak area). Elemental Analysis—C₅₀H₈₂N₄O₈, 4HCl,FW=1013.05, calculated: C, 59.28; H, 8.56; N, 5.53. Found: C, 58.94; H,8.21; N, 5.43. Chlorine elemental analysis by titration method(anhydrous basis): theory: 14.00%. found: 14.05%.

¹H NMR (D₂O, 400 MHz): δ=0.72 (d, J=6.4 Hz, 6H, 2×CH %), 1.32-1.42 (m,16H) 1.62-1.68 (m, 2H, 2/CH), 2.16 (dd, J=13.4, 9.2 Hz, 2H, 2×ArCH),2.72 (dd, J=13.2, 4.6 Hz, 2H, 2×ArCH), 2.41-2.59 (m, 16H, 8× morpholineCH₂), 2.70 (t, J=12.2 Hz, 8H, 4×CH₂N), 3.46-3.72 (m, 16H, 8× morpholineCH₂), 4.01 (t, J=5.6 Hz, 8H, 4×CH₂O), 6.57-6.70 (m, 6H, 6′ArH) ppm;consistent with structure.

¹³C NMR (D₂O, 100 MHz): δ=15.96, 23.91, 24.89, 38.57, 39.08, 53.31,53.79, 57.46, 59.79, 66.50, 66.58, 66.70, 113.68, 114.69, 121.39,134.75, 146.39, 148.29 ppm; consistent with structure.

MS (FAB) m/e: 867 (M+), 434 (½M+), consistent with free base structure(C₅₄H₈₂N₄O₈).

Example 9 Synthesis of1,4-bis{3,4-bis[4-(N-methyl-piperazino-N′-yl)butoxyl]phenyl}-2,3-dimethyl-(2R,3S)-butanefree base (C₅₄H₉₄N₈O₄, FW=919.38) “Compound N”; octa-hydrochloride salt(C₅₄H₉₄N₈O₄.8HCl, FW=1211.06) “Compound O” Step 1: Synthesis ofN-methyl-N′-(4-chlorobutyl)piperazine

In a 2 L three-necked round bottomed flask was added N-methyl-piperazine(100.16 g, 1.0 mol), acetone (1000 mL), and anhydrous potassiumcarbonate (276.42 g, 2.0 mol, 2.0 equiv.). The flask was equipped with amechanical stirrer and a condenser. 4-chloro-1-bromo-butane (188.6 g,1.1 mol, 1.1 equiv.) was added dropwise under continuous stirring atroom temperature over a period of 30 min. The suspension mixture wasthen stirred at 40° C. The progress of the reaction was monitored byTLC, which confirmed the reaction was completed after 4 hours. Themixture was cooled to room temperature and the insoluble materials wereremoved by filtration and washed with dichloromethane (2×100 mL). Thecombined filtrates were concentrated under vacuum until they became dry.The residue was then mixed with dichloromethane (500 mL). Insolublematerials were removed by filtration and washed by dichloromethane(2×100 mL). The combined filtrate and washings were concentrated andpurified through a flash silica gel column, which gave the expectedproduct as light yellow oil. (92.5 g, 48.5% yield).

¹NMR (CDCl₃, 300 MHz), 8=1.37 (m, 2H, CH₂), 1.81 (m, 2H, CH₂), 2.25 (s,3H, NCH), 2.33 (m, 8H, 4 NCH₂), 3.10 (t, J=6.5 Hz, 2H, 2CH₂N), 3.65 (t,J=6.8 Hz, 2H, CH₂Cl) ppm; consistent with the structure.

¹³C NMR (CDCl₃, 75 MHz), δ=25.4, 25.9, 44.1, 45.8, 52.1, 53.1, 55.8 ppm;consistent with the structure.

MS (EI), m/e=191 (M+1); consistent with the structure.

Step 2: Synthesis of1,4-bis{3,4-bis[4-(N-methyl-piperazino-N′-yl)butoxyl]phenyl}-2,3-dimethyl-(2R,3S)-butane“Compound N”

To a solution containing NDGA (602 mg, 2.0 mmol) and lithium hydroxidemonohydrate (1.0 g, 24.0 mmol, 12 equiv.) in tert-butanol (100 mL) wasadded N-methyl-N-(4-chlorobutyl)piperazine (2.29 g, 12.0 mmol. 6.0equiv.). The solution was heated at 50° C. under continuous stirring.The reaction was monitored by TLC(CH₂Cl₂:McOH:Et₃N=92:6:2. V/V/V), whichconfirmed the reaction was completed after 20 hours. The reactionsuspension was cooled to room temperature, and partitioned withdichloromethane (200 mL) and water (100 mL). The mixture was shaken tomix well. The organic phase was separated and the aqueous phase wasextracted with dichloromethane (2×100 mL). The organic layer andextracts were combined and washed with aqueous saturated sodiumbicarbonate (100 mL), and brine (10 mL). After drying over anhydroussodium sulfate, the solution was concentrated under reduced vacuum. Theresidue was purified through a silica gel column using dichloromethane,methanol and triethylamine (92:6:2, V/V/V) as an eluent, which gave theexpected product as white semi-solid material (855.02 mg, 46.5% yield).

HPLC purity: 98.2% (% peak area). Elemental Analysis—C₅₄H₉₄O₈N₄,FW=919.38. Calculated: C, 70.55; H, 10.31; N, 12.19. Found: C, 70.65: H,10.11: N, 12.43: consistent with structure.

¹H NMR (CDCl₃, 400 MHz): δ=0.76 (d, J=6 Hz, 6H, 2×CH₃), 1.36-1.56 (m,16H), 1.65-1.71 (m, 4H), 2.42 (s, 6H), 2.45-2.65 (m, 22H), 2.85 (t,J=6.0 Hz, 4H), 2.95 (t, J=6.0 Hz, 4H), 4.23 (q, J=6.0 Hz, 8H), 6.65-6.78(m, 6H, 6×ArH) ppm; consistent with structure.

¹³C NMR (CDCl₃, 100 MHz): δ=15.61, 23.55, 24.56, 32.26, 35.58, 46.27,53.13, 55.05, 56.36, 66.44, 66.86, 113.77, 115.63, 121.15, 145.66,147.97, 150.02 ppm; consistent with structure.

MS (FAB) m/e: 920 (M+), 460 (M+), consistent with the structure.

Step 3: Synthesis of1,4-bis{3,4-bis[4-(N-methyl-piperazino-N′-yl)butoxyl]phenyl}-2,3-dimethyl-(2R,3S)-butaneocta-hydrochloride “Compound O”

To an ice cooled (0-5° C.) solution of aqueous concentrated HCl (2.2 mLof 11 N HCl, 24 mmol. 24 mole equiv.) in 95% ethanol (7 mL) was addeddropwise a solution of 1,4-bis{3,4-bis[4-(N-methyl-piperazino-N′-yl)butoxy]phenyl}-2,3-dimethyl-(2R,3S)-butane(919.33 mg, 1.0 mmols) in 95% ethanol (7 mL). The solution was allowedto stir at 0-5° C. for three hours and the solvent was removed on arotary evaporator while keeping the temperature of the water bath at 45°C. The hydrochloride salt was dried under high vacuum for 48 hours. Thecrude product was then crystallized from ethanol:ether to give 914.35 mgof the product (75.5% yield) after drying under high vacuum for 72hours. The analytical data for this product are given below.

m.p. 215-220° C. (dec.).

HPLC purity: 98.2% (% peak area). Elemental Analysis—C₅₄H₉₄N₈O₄.8HCl,FW=1211.06 calculated: C, 53.55: H, 8.49: N, 9.25. Found: C, 53.24; H,8.21; N, 9.43. Chlorine elemental analysis by titration method(anhydrous basis): theory: 23.42%. found: 23.35%; consistent with thestructure.

¹H NMR (D₂O, 400 MHz): δ=0.75 (d, J=6 Hz, 6H, 2×CH₃), 1.33-1.46 (m,16H), 1.55-1.64 (m, 4H), 2.32 (s, 6H), 2.35 (a, 6H), 2.45-2.65 (m, 16H),2.83 (t, J=6.0 Hz, 4H), 2.82 (t, J=6.0 Hz, 4H), 4.10 (q, J=6.0 Hz, 8H),6.65-6.78 (m, 6H, 6×ArH) ppm; consistent with the structure. 9 (Ar),132.8 (Ar), 146.4 (Ar), 148.1 (Ar) ppm; consistent with structure.

¹³C NMR (D₂O, 100 MHz): δ=15.91, 23.85, 25.06, 31.26, 35.38, 46.17,53.73, 55.15, 57.36, 67.34, 67.46, 114.47, 114.73, 120.95, 146.06,148.77, 149.92 ppm; consistent with the structure.

MS (FAB) m/c: 920 (M+), 460 (M+); consistent with the structure.

The following Prophetic Examples 1-5 show reaction schemes that arebelieved to be effective to make the indicated compounds.

Prophetic Example A Synthesis of1,4-bis{3,4-bis[2-(1-methyl-piperazin-4-yl)-ethoxy]phenyl}-2,3-dimethyl-(2R,3S)-butanefree base (C₄₆H₇₈N₈O₄; FW=807.16); tetrakis-hydrochloride salt(C₄₆H₇₈N₈O₄.4HCl, FW 953.01)

Prophetic Example B Synthesis of1,4-bis{3,4-bis[2-(piperidin-1-yl)ethylcarbamoyloxy]phenyl}-2,3-dimethyl-(2R,3S)-butanefree base (C₅₀H₇₈N₈O₈, FW=919.20); tetrakis-hydrochloride salt(C₅₀H₇₈N₈O₈.4HCl, FW=1065.05)

Method 1

Method 2

Prophetic Example C Synthesis of1,4-bis{3,4-bis[2-(morpholin-1-yl)ethylcarbamoyloxy]phenyl}-2,3-dimethyl-(2R,3S)-butanefree base (C₄₆H₇₀N₈O₁₂, FW=927.09); tetrakis-hydrochloride salt(C₄₆H₇₀N₈O₁₂.4HCl, FW=1072.94)

Prophetic Example D Synthesis of1,4-bis{3,4-bis[(2-N,N-dimethylaminoethyl)carbamoyloxy]phenyl}-2,3-dimethyl-(2R,3S)-butanefree base (C₃₈H₆₂N₈O₈, FW=758.95)

Prophetic Example E Synthesis of1,4-bis{3,4-bis[(furan-2-yl)methyl-carbamoyloxy]phenyl}-2,3-dimethyl-(2R,3S)-butanefree base (C₄₂H₄₂N₄O₁₂, FW=794.80)

The invention will further be described with respect to the followingspecific, non-limiting working examples relating to in vitrocytotoxicity and effectiveness studies, following the general protocolsthat were performed.

Cytotoxicity and Effectiveness Studies

Certain of the compounds of the present invention have been studied invitro. The in vitro studies have established that the various classes ofthe NDGA derivatives of the present invention would be safe andeffective for prophylactic or after-onset treatment of a viral infectionor a proliferative, inflammatory, metabolic or vascular disease. Thefollowing examples explain the studies involved in such testing.

Cytotoxicity Studies

Studies performed regarding cytotoxicity included the well-known MTS,Trypan Blue and MTT protocols. The MTS studies were done using theCellTiter 96®AQ_(ueous) One Solution Cell Proliferation Assay (PromegaCorporation, Madison, Wis. USA). Metabolically active, namely viable,cells turn MTS, a tetrazolium compound(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt) into colored formazan, which is soluble in tissue culturemedium. The measurement of the absorbance of the formazan is read at 490nm. The ready to use reagent is added directly to the cells in media in96-well plates, incubated 1-4 hours and the results are recorded by theplate reader. The IC₅₀ is estimated by graphing the gathered data. TheIC₅₀ is the concentration of the tested material that inhibits 50% ofgrowth or viability of the tested material compared to a control.

In the Trypan Blue assay, cells are trypsinized and a sample is added toa solution of trypan blue dye and saline. Viable cells are able to keepthe dye on the outside of their membrane, damaged or dead cells are not.Viable and non-viable (blue) cells are counted, and the percent viableis calculated. The proliferation rate is calculated using the valuesfrom placebo treated cells and compares the viability of treated cellsto that of non-treated cells.

The MTT assay is a colorimetric method for determining the number ofviable cells. Metabolically active cells turn the MIT reagent,(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, intopurple-colored formazan crystals that are solubilized in dimethylsulfoxide (DMSO). The MTT reagent is added to MTT (colorless) media. Themedia is added to the cells and they are incubated for 4 hours. DMSO isadded to the cells and mixed. The colored solutions are then read at 540nm and corrected at 630 nm. The IC₅₀ is estimated by graphing thegathered data.

Antiviral Activity SEAP Assay

Antiviral activity is determined using a SEAP (SEcreted AlkalinePhosphatase) assay, in which cells are co-transfected with SEAP and TATplasmids. TAT is a transactivator, human immunodeficiency virus (HIV)gene expression and is one of the two or more necessary viral regulatoryfactors (TAT and REV) for HIV gene expression. TAT acts iv binding tothe TAR RNA element and activating transcription from the long terminalrepeat (LTR) promoter. The TAT protein stabilizes elongation oftranscription and has also been shown to be involved is transcriptioninitiation. Previous studies have shown that TAT mediates reduction ofantibody-dependent T cell proliferation, contributing substantially tothe failure of the immune response. TAT also directly stimulatesKaposi's cell growth.

Since TAT has no apparent cellular homologs, this strong positiveregulator has become an attractive target for the development ofanti-AIDS drugs. In contrast to currently available HIV reversetranscriptase inhibitors (AZT, DDI) or potential protease inhibitorsthat prevent new rounds of infection, an inhibitor which suppressesviral gene TAT regulated expression of integrated proviral DNA willarrest the virus at an early stage (Hsu et al., Science 254:1799-1802,1991). Efforts aimed at the elucidation of factors which control geneexpression at transcriptional and post-transcriptional levels in hosteukaryotes have recently made possible quantitative assessment of TATfunction (Sim, Ann. N.Y. Acad. Sci. 616: 64-70, 1990). To screen forinhibitors for TAT regulated transactivation (TAT-TRS), the SEAPreporter gene is put under the control of HIV-1 LTR promoter in theplasmid pBC12/HIV/SEAP. The TAT-coded activity is supplied by a secondplasmid construct pBC₁₂/CMV/t2. Transient co-transfection of COS-7 cellswith these two plasmids leads to secretion of alkaline phosphatase intothe culture medium which is analyzed by a simple colorimetric assay(Berger et al., Gene 66: 1-10, 1988). The SEAP assay, therefore,provides an indirect determination of TAT transactivation.

In the SEAP assay, an inhibitor should cause reduction of SEAP reportergene expression via transactivation of the HIV-1 LTR promoter by TATprotein (TAT-TRS). The TAT protein is expressed under the control of acytomegalovirus promoter and induces the expression of thenon-endogenous, heat resistant form of secreted alkaline phosphatase. IfTAT transactivation is blocked by a drug, the reporter SEAP will not beexcreted into the media. SEAP is detected colorimetrically by thepara-nitrophenyl phosphate substrate at 405 nm. See Gnabre¹³. The cellsare analyzed by the MTT assay to compare cytotoxicity to SEAPinhibition. The goal is for the drug to inhibit SEAP without being tootoxic.

Antiproliferative Activity

Antiproliferative activity is determined using the TiterTACS® ApoptosisDetection Kit (R&D Systems Inc., Minneapolis, Minn. USA) relating toapoptosis of cells based on DNA fragmentation, and by the ELISA VEGF(vascular epithelial growth factor) and survivin assays.

In the DNA fragmentation assay, in situ detection of apoptosis isspecifically achieved with TiterTACS® 96-well Apoptosis Detection kit,Catalog No. TA600 (R&D Systems, Inc.). The TiterTACS® assay providesquantification of apoptosis in cultured cells without direct counting oflabeled cells using colorimetric detection. Cells are treated with thetest compounds, left untreated as experimental negative controls, ortreated with TACS nuclease as positive controls. TACS nuclease allowspositive controls to be generated for each experimental system: a brieftreatment of cells with the TACS nuclease prior to labeling generatesDNA breaks in every cell, providing an appropriate positive controlspecific for the system under study. The TdT enzyme that catalyzes theaddition of dNTPs to DNA fragments allows for colorimetric detection ofat 450 nm by using a streptavidin-HRP solution followed by theTACS-Sapphire substrate. A high absorbance at 450 nm is indicative ofapoptosis in the cells. Treated cells are compared to untreated cellsand to nuclease-cleaved cells to assess the extent of apoptosis.

ELISA (Enzyme-Linked ImmunoSorbent Assay) VEGF studies were donefollowing manufacturer's instructions using the Endogen® Human VEGFELISA kit, Catalog No. EHVEGF (Pierce Biotechnology, Inc., Rockford,Ill. USA). Hypoxic conditions are created for the cells by treating themwith desferrioxamine (DFO), which chelates iron and causes the cells tosecrete VEGF into the media. The supernatant (media) was removed andfrozen for testing, and the cells were counted with the Trypan Blueassay so that the results can be normalized to cell count. The VEGF ismeasured with a sandwich ELISA that captures the protein in media on anantibody-coated microplate, and then uses a biotinylated antibodyreagent to detect the protein. Results from a standard curve enable theuser to quantify the amount of protein in each sample.

ELISA-Survivin Assay

Survivin is an inhibitor of apoptosis that is abundantly expressed inmany human cancers, but not in normal adult human tissue, and isconsidered a possible modulator of the terminal effector phase of celldeath/survival. Survivin is expressed in G₂-M in a cell cycle-dependentmanner, binding directly to mitotic spindle microtubules. It appearsthat survivin phosphorylation on Thr34 may be required to maintain cellviability at cell division, and expression of aphosphorylation-defective survivin mutant has been shown to triggerapoptosis in several human melanoma cell lines. Phosphorylated survivinacts on the caspase pathway to suppress the formation of caspase-3 andcaspase-9, thereby inhibiting apoptosis. Thus, compounds that reduce theexpression of survivin will be expected to increase the rate ofapoptosis and cell death.

Effects of the tested compounds on survivin were studied followingmanufacturer's instructions using the Surveyor™ IC Human Total SurvivinImmunoassay, Catalog No. SUV647 (R&D Systems, Inc.). Cell lysates wereanalyzed for survivin protein content. The kit includes a plate coatedwith an antibody specific for survivin and a biotinylated antibodyreagent that recognizes survivin bound to the plate. The plate is readon a plate reader set at 450 nm and corrected at 540 nm. A protein assayaccording to the Bradford method (Bradford, M. 1976, Anal Biochem 72:248-254) was used to quantify and normalize the samples according tototal protein content. Results from a standard curve enable the user toquantify the amount of survivin protein in each sample.

Anti-Inflammatory Activity

Anti-inflammatory activity was determined based on the effect of testedcompounds on primary human keratinocytes (PHKs). PHKs play an importantrole in inflammatory processes, synthesizing a number of cytokines,adhesion molecules and growth factors. Studies were conducted todetermine whether tested compounds could inhibit keratinocytes toprevent or reduce production of interferon gamma (IFN-γ), interleukin-8(IL-8), tumor necrosis factor alpha (TNF-α), granulocyte/macrophagecolony-stimulating factor (GM-CSF), intercellular adhesion molecule-1(ICAM-1, also known as CD54) and monocyte chemotactic protein-1 (MCP-1).PHKs are first treated with TNF-α to induce a pro-inflammatory state andrelease of pro-inflammatory cytokines. Specific cytokines are thenassayed following manufacturer's instructions using R&D Systems. Inc.'sprotein assays (Quantikine ELISA kits; Catalog Nos. DCP00 for MCP-1 kit,DCM00 for GM-CSF kit, and DIF50 for IFN-γ).

General Protocols for Cytotoxicity Studies Relating to Antiviral andAntiproliferative Activity

Three cell lines obtained from ATCC and maintained as directed by ATCCwere tested: HeLa (cervical adenocarcinoma), A549 (lung carcinoma), andCOS-7 (SV40 transformed monkey kidney). Studies of these cell lines areconsidered to be indicative of the effect of tested substances onmammalian, including human, diseases.

All compounds were dissolved in DMSO and DMSO is used as the placebo.The samples were first dissolved into 10 mM dilutions in DMSO and thendiluted further to 5, 1, 0.5, 0.1, 0.05, and 0.01 mM solutions. Thesesolutions were added to the media at a 1% concentration (1 μl to 100 μlmedia) to treat the cells with 100, 50, 10, 5, 1, 0.5, and 0.1 μM of thecompound. The compounds that were found to have low IC₅₀s were dilutedfurther in DMSO. Solutions were checked before use for precipitation,especially the 10 mM solutions. If there was precipitation, they werewarmed 65° C. and added to warmed media.

Wells on a 96-well plate were seeded with 1.5-8×10⁴ cells in 100 μlmedia and incubated overnight. The outside wells of the plate werefilled with 200 μl sterile, deionized (DI) water to curb mediaevaporation. After 24 hours, test chemicals were prepared in media at aconcentration of 1% media volume. The test sample media were added,mixing with pipette before adding 100 μl per well. Media was added tothe “Media Only” wells. The well plate and its contents were incubatedfor 24-72 hours, depending on the study conducted. On the thy of the MTSassay, the MTS reagent was removed from the refrigerator and brought toroom temperature. Using the multichannel pipette, 20 μl of the MTSreagent was added to each well and incubated for one to four hours. Theplate was read at 490 nm with a reference wavelength of 690 nm after onehour in the incubator thereafter until the blank wells were at about 0.2OD. The results were then scanned into a Microsoft® Excel® templatedesigned to perform all necessary calculations whim data are entered.The data were checked for statistical errors; data points that arewithin 10% of the mean of the data points for that group were included.The average of the blanks (“Media Only”) were subtracted and the datawere inserted into a chart that represents growth response,treated/untreated.

SEAP Protocol

The following SEAP protocol was used as a reporter system for measuringthe activity of TAT-mediated transactivation of HIV transcription. TheMTT assay measures cellular-proliferation and is used to verify that thelevels of SEAP activity are not solely due to cytotoxicity. These assayswas used in screening for potential drug compound leads as antiviralagents, and particularly, anti-HIV candidates.

COS-7 (green monkey kidney) cells were co-transfected using Fugene 6reagent (Roche Applied Science, Cat. No. 11815091001, Indianapolis,Ind., USA) with two plasmids: pHIVSEAP (SEAP expression vector under thecontrol of the HIV LTR promoter) and pCTAT (HIV TAT transcription factorexpression vector under the control of a CMV (cytomegalovirus)promoter). After test compound treatment for 48 hours, samples wereanalyzed for SW activity at 405 nm after addition of the p-nitrophenylphosphate substrate. The percentage of SEAP activity inhibition wascalculated in relation to the placebo control.

Example 10 SEAP/MTT and MTS Studies—Cytotoxicity and AntiviralEffectiveness

The results of the SEAP/MTT and MTS studies on the indicated compoundsfrom the working Examples above based on the general protocols set forthabove are set forth in the following Table 3, where the left hand columnidentifies the compound tested and EC₅₀ indicates the concentration ofthe compound having 50% of the effect of the control. ICs, was definedabove.

TABLE 3 SEAP COS-7 Compound EC₅₀ IC₅₀ A549 IC₅₀ HeLa IC₅₀ A 0.5-1 μM0.7-0.9 μM 0.8-0.9 μM 2-3 μM B 1-5 μM D 1-5 μM 5-10 μM M 1-5 μM 5-10 μMO 8-9 μM 10-30 μM F Variable Variable >100 μM >100 μM Response ResponseG in H₂O 15-20 μM 15-20 μM H in H₂O 15-20 μM 15-20 μM I 60-80 μM 70-80μM 65-70 μM 20-30 μM K >100 μM >100 μM J 80-90 μM 80-90 μM 80-90 μM >100μM

Results from compounds with biological activity in two cell lines testedusing the MTS assay as an indicator of cell viability are shown in Table3. IC₅₀ concentrations show various degrees of inhibition of cellviability by these compounds. All compounds were able to reduce cellviability, and therefore, induce apoptosis, in a dose dependent manner.Compound A showed the strongest cell viability inhibition in A549 cells,while its hydrochloride salt (Compound B) showed the strongestinhibition in HeLa cells.

The extent of inhibition of TAT-transactivation as determined by SEAPassay also varied among compounds, as also shown in Table 3. Compound Aalso showed significant antiviral activity with EC₅₀ around 0.5-1.0 μM.Inhibitory concentrations of cell viability in the test cells (COS-7) asdetermined by MTT were usually similar as the effective concentrationsfor TAT-transactivation.

Example 11 Antiproliferative Activity Based on TiterTACS®, VEGA andSurvivin Aptoptosis Studies

The results of the TiterTACS® DNA fragmentation, ELISA VEGA and ELISAsurvivin apoptosis studies on the indicated compounds from the workingExamples above, based on the general protocols above, are set forth inthe following Table 4.

TABLE 4 DNA Survivin Compound fragmentation IC₅₀ VEGF IC₅₀ B Negative  2μM D Positive  4 μM  9 μM M Positive  5 μM  9 μM O Positive 14 μM 18 μMG in H₂O 25 μM H in H₂O Positive 10 μM 23 μM Positive = inducesapoptosis Negative = does not induce apoptosis

The compounds tested have shown significant reduction of survivinprotein levels at low micromolar concentrations. Consequently, they havealso shown strong apoptosis indication as determined by the DNAfragmentation assay. Compounds D, M, O and H reduce survivin proteinexpression and are therefore able to induce apoptosis. These compoundsare able to decrease survivin and induce apoptosis in a dose-dependentmanner. Compound D was the most potent inhibitor of survivin proteinproduction with IC₅₀ for survivin production around 4 μM.

Release of VEGF protein is also decreased by these compounds. Inhibitionof VEGF protein production ranged in the low micromolar concentrationsfor compounds B, D, M, O, G and H. Inhibition by all compounds wasobserved in a dose-dependent fashion. Compound B showed an IC₅₀ for VEGFproduction around 2 μM, while compounds D and M both showed VEGF IC₅₀ ataround 9 μM.

The results from Table 4 indicate that the compounds of the presentinvention are effective in inhibiting viral activity and causing celldeath (apoptosis) in the cell lines rested. Of the compounds tested,Compounds A, B, D, G, H, M and O appear to be more effective than theothers due to their lower ECs, and IC; values. Compound A appears to bethe most effective.

Example 12 U.S. National Cancer Institute DTP Human Tumor Cell LineScreen

The United States National Cancer Institute (NCI) provides adevelopmental therapeutics program (DTP)(http://dtp.nci.nih.gov/branches/btb/ivelsp.html) to screen submittedsubstances in support of cancer drug discovery. The In Vitro Cell LineScreening Project (IVCLSP) is a dedicated service providing directsupport to the DTP anticancer drug discovery program and is designed toscreen up to 3,000 compounds per year for potential anticancer activity.The operation of this screen utilizes 60 different human tumor celllines, representing leukemia, melanoma and cancers of the lung, colon,brain, ovary, breast, prostate, and kidney. The aim is to prioritize forfarther evaluation, synthetic compounds or natural product samplesshowing selective growth inhibition or cell killing of particular tumorcell lines. This screen is unique in that the complexity of a 60 cellline dose response produced by a given compound results in a biologicalresponse pattern which can be utilized in pattern recognition algorithms(COMPARE program. See:http://dtp.nci.nih.gov/docs/compare/compare.html). Using thesealgorithms, it is possible to assign a putative mechanism of action to atest compound, or to determine that the response pattern is unique andnot similar to that of any of the standard prototype compounds includedin the NCI database. In addition, following characterization of variouscellular molecular targets in the 60 cell lines, it may be possible toselect compounds most likely to interact with a specific moleculartarget.

The screening is a two-stage process, beginning with the evaluation ofall compounds against the 60 cell lines at a single dose of 10 μM. Theoutput from the single dose screen is reported as a mean graph and isavailable for analysis by the COMPARE program. Compounds which exhibitsignificant growth inhibition are evaluated against the 60 cell panel atEve concentration levels.

Methodology of the In Vitro Cancer Screen

The human tumor cell lines of the cancer screening panel are grown inRPMI 1640 medium containing 5% fetal bovine serum and 2 mM L-glutamine.For a typical screening experiment, cells are inoculated into 96 wellmicrotiter plates in 100 L at plating densities ranging from 5.000 to40,000 cells/well depending on the doubling time of individual celllines. After cell inoculation, the microtiter plates are incubated at370° C., 5% CO₂, 95% air and 100% relative humidity for 24 hours priorto addition of experimental drugs.

After 24 hours, two plates of each cell line are fixed in situ withtrichloroacetic acid (TCA), to represent a measurement of the cellpopulation for each cell line at the time of drug addition (Tz).Experimental drugs are solubilized in DMSO at 400-fold the desired finalmaximum test concentration and stored frozen prior to use. At the timeof drug addition, an aliquot of frozen concentrate is thawed and dilutedto twice the desired final maximum test concentration with completemedium containing 50 μg/ml gentamicin. Additional four, 10-fold or ½ logserial dilutions are made to provide a total of five drug concentrationsplus control. Aliquots of 100 μl of these different drug dilutions areadded to the appropriate microtiter wells already containing 100 μl ofmedium, resulting in the required final drug concentrations.

Following drug addition, the plates are incubated for an additional 48hours at 37° C., 5% CO₂, 95% air, and 100% relative humidity. Foradherent cells, the assay is terminated by the addition of cold TCA.Cells are fixed in situ by the gentle addition of 50 μl of cold 50%(w/v) TCA (final concentration, 10% TCA) and incubated for 60 minutes at4° C. The supernatant is discarded, and the plates are washed five timeswith tap water and air dried. Sulforhodamine B (SRB) solution (100 μl)at 0.4% (w/v) in 1% acetic acid is added to each well, and plates areincubated for 10 minutes at room temperature. After staining, unbounddye is removed by washing five times with 1% acetic acid and the platesare air dried. Bound stain is subsequently solubilized with 10 mM trizmabase, and the absorbance is read on an automated plate reader at awavelength of 515 nm. For suspension cells, the methodology is the sameexcept that the assay is terminated by fixing settled cells at thebottom of the wells by gently adding 50 μl of 80% TCA (finalconcentration, 16% TCA). Using the seven absorbance measurements [timezero, (Tz), control growth, (C), and test growth in the presence of drugat the five concentration levels (Ti)], the percentage growth iscalculated at each of the drug concentrations levels. Percentage growthinhibition is calculated as:

[(Ti−Tz)/(C−Tz)]×100 for concentrations for which Ti>/=Tz

[(Ti−Tz)/Tz]100 for concentrations for which Ti<Tz.

Three dose response parameters are calculated for each experimentalagent. Growth inhibition of 50% (GI₅₀) is calculated from[(Ti−Tz)/(C−Tz)]100=50, which is the drug concentration resulting in a50% reduction in the net protein increase (as measured by SRB staining)in control cells during the drug incubation. The drug concentrationresulting in total growth inhibition (TGI) is calculated from Ti=Tz. TheLC₅₀ (concentration of drug resulting in a 50% reduction in the measuredprotein at the end of the drug treatment as compared to that a thebeginning) indicating a net loss of cells following treatment iscalculated from [(Ti−Tz)/Tz]×100=−50. Values are calculated for each ofthese three parameters if the level of activity is reached; however, ifthe effect is not reached or is exceeded, the value for that parameteris expressed as greater or less than the maximum or minimumconcentration tested.

A summary of the results of the NCI DTP study as described above forCompound A at 10 μM is set forth in Table 5:

TABLE 5 Compound A Cell Line Panel Mean Growth (# cell lines) Percent at10 μM NSCLC (8) −90.92 Colon Cancer (7) −94.08 Breast Cancer (6) −67.95Ovarian Cancer (6) −85.89 Leukemia (6) −81.93 Renal Cancer (6) −95.54Melanoma (8) −92.38 Prostate (2) −95.47 CNS (6) −93.97

The results indicate that Compound A at 10 μM concentration hadbroad-based activity against a number of the types of the 60 cell lines.The most inhibitory effects were shown against the renal cancer celllines, followed closely by prostate cancer cell lines, with the leastinhibitory effects shown against breast cancer.

Example 13 Anti-Inflammatory and Anti-Vascular Disease Activity PHKStudies

Anti-inflammatory activity of several of the compounds of the previouslydescribed working examples was determined based on the effect of testedcompounds on primary human keratinocytes (PHKs) as described above andfollowing the manufacturer's instructions for the various kits used inthe studies. While these studies more typically are used mostly foranti-inflammatory investigations, they can also apply to vasculardiseases, since the results in keratinocytes may be extrapolated tovascular endothelium.

Only MCP-1 data with PHKs is dose-dependent, so an IC₅₀ for MCP-1 can becalculated, but not for the other cytokines. Results of the MCP-1studies on various compounds are shown in the following Table 6.

TABLE 6 MCP-1 Compound IC₅₀ B  2 μM F >100 μM  I 25 μM J 70 μM

Results from compounds with anti-inflammatory activity in TNF-α-treatedPHK as assayed by MCP-1 protein production are shown in Table 6. IC₅₀concentrations for MCP-1 protein show various degrees of inhibition ofthis inflammatory cytokine. In results of other of these tests, allcompounds that were tested were able to reduce MCP-1 protein productionin a dose dependent manner. Compound B showed the strongest inhibitionof MCP-1 in TNF-α-treated PHK.

Example 14 Permeability Studies as Indicative of Oral Bioavailability

Permeability studies were conducted by an outside contractor on behalfof the assignee of the present invention and application to determinethe permeability of Compound A through Caco-2 monolayers. An importantfactor of oral bioavailability is the ability of e compound to beabsorbed in the small intestine. Measurement of drug apparentpermeability (P_(app)) through cell monolayers is well correlated withhuman intestinal absorption, and several mammalian cell lines, includingCaco-2, LLC-PK1 and MDCK, are appropriate for this measurement(Artursson, P. et al., Correlation between oral drug absorption inhumans and apparent drug permeability coefficients in human intestinalepithelial (Caco-2) cells. Biochem Biophys Res Comm 175:880-885 (1991);Stewart. B. H., et al., Comparison of intestinal permeabilities inmultiple in vitro and in situ models: relationship to absorption inhumans. Pharm Res 12:693 (1995)). P-Glycoprotein (P-gp, encoded by MDR1)is a member of the ABC transporter super family and is expressed in thehuman intestine, liver and other tissues. Intestinal expression of P-gpmay affect the oral bioavailability of drug molecules that aresubstrates for this transporter. Interaction with P-gp can be studiedusing direct assays of drug transport in polarized cell systems such asCaco-2 cell monolayers, and human P-gp cDNA-expressing LLC-PK₁ and MDCKcell monolayers.

Caco-2 cells (human adenocarcinoma colonic cell line Caco-2, ATCC Cat.No. HTB-37, used between passages 18 and 45) were seeded onto BD Falcon™HTS 24-Multiwell, 1 μm culture inserts (BD catalog #351180) (BDBiosciences Discovery Labware, Woburn, Mass., USA.). The cells werecultured for 21-25 days with media replacement every 3-4 days. Monolayerintegrity was evaluated by pre-experimental trans-epithelial electricalresistance (TEER) measurements and post-experimental lucifer yellow A toB flux determinations.

Transport buffer was HBSS (Hanks Balanced Salt solution) buffered withthe addition of 10 mM HEPES(N-[2-Hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]), and pHadjusted to 7.4 with NaOH. Receiver solution was prepared by adding 1%DMSO to transport buffer. The test solution was of Compound A in DMSO intransport buffer at a final DMSO concentration of 1%. Donor solutionsfor two permeability comparator compounds (50 M [³H]-propranolol as“High” and 50 μM [¹⁴C]-mannitol as “Low”) as well as a positive controlP-gp substrate (5 μM [³H]-digoxin) were prepared by diluting aliquots ofradiolabeled and non-radiolabeled stock solutions into transport bufferat a final DMSO concentration of 1%.

The test articles were assayed at a single concentration (1 μM) in bothA to B and B to A directions. The donor and receiver solutions wereadded to the apical or basolateral chambers of the monolayers (dependingon the direction of transport to be measured). The monolayers wereincubated on an orbital shaker (50 rpm) at 37° C., with ambient humidityand CO₂. After 90 minutes, samples from the donor and receiver chamberswere removed for analysis.

To determine the extent of non-specific binding of the sample to theassay plate, the sample solution was incubated under the conditionsdescribed above in a single well of a 24-well assay plate. After 90minutes, the sample was removed from each well for analysis. After allsamples were collected, lucifer yellow solution was added to eachmonolayer at a final concentration of 100 μM. The inserts were placed ina new receiver plate containing transport buffer. After a 30 min.incubation on an orbital shaker (50 rpm) at 37° C., with ambienthumidity and CO₂, samples were removed from the receiver chamber tomeasure percent lucifer yellow flux.

Two permeability comparator compounds representing high permeability (50μM [³H]-propranolol) and low permeability (50 μM [¹⁴C]-mannitol) wereassayed in the A to B direction, with samples from the donor andreceiver chambers taken at one time point of 90 minutes. The controlP-gp substrate was 5 μM [³H]-digoxin assayed in both A to B and B to Adirections, with samples from the donor and receiver chambers taken atone time point of 90 minutes. Duplicate monolayers were used for eachincubation.

Samples were analyzed by LC/MS/MS using peak area ratios to an internalstandard. Test article concentrations were calculated based on the peakarea ratios obtained for appropriate dilutions of the donor solutioncontaining the test article at the nominal concentration in transportbuffer. Samples were diluted as appropriate to ensure that the responsewas within the linear range of the mass spec detector. Comparator andcontrol samples were analyzed by liquid scintillation counting. Luciferyellow concentrations were determined using a fluorescence plate reader.

Pre-experimental trans-epithelial electrical resistance (TEER)measurements (mean 411 Ωcm²) and post-experimental lucifer yellow A to Bflux (mean 0.1%) confirmed monolayer integrity. The polarization of thepositive control digoxin confirmed a functioning P-gp transport model.The P_(app) values for the permeability comparators mannitol andpropranolol were within historical ranges observed at the testingfacility with this test system (mean 4.1×10⁻⁷±SD 2.4×10⁻⁷ for mannitol,1.5×10⁻⁵±4.2×10⁻⁵ for propranolol) indicating a properly functioningmodel.

The results for the permeability comparators mannitol and propranolol aswell as for the control P-gp substrate digoxin are reported in thefollowing Table 7.

TABLE 7 P_(app) [cm/s] Polariza- Mass balance mean tion Ratio mean A toB to mean A to B to Compound B A (B-A/A-B) B A digoxin (5 μM) 2.28E−061.24E−05 5.4 73% 79% propranolol (50 μM) 1.42E−05 n.d. n.d. 71% n.d.mannitol (50 μM) 5.14E−07 n.d. n.d. 96% n.d. n.d.—not determined

Bidirectional permeability data as well as polarization ratios obtainedfor the test compound in Caco-2 cell monolayers are reported in Table 8.

TABLE 8 Nominal conc. P_(app) [cm/s] Polarization Ratio Test article[μM] A to B B to A [B-A/A-B] Compound A 1.0 9.75E−06 9.61E−06 1.74E−061.72E−06 0.18

The recovery of Compound A from the apical and basolateral chambers(mass balance) as well as from a single well of a 24-well culture platewithout cells (non-specific binding) at the end of the incubation isreported in Table 9.

TABLE 9 Non- specific Binding Nominal conc. Mass Balance * % Testarticle [μM] A to B B to A Recovery Compound A 1.0 36% 39% 38% 30% 13% *Mass balance/recovery values of >100% are typically due to increasedsolubility over the course of the incubation

CONCLUSIONS

The results were very encouraging. Permeability studies in Caco-2 cellmonolayer as an in vitro model of intestinal absorption demonstrate thatCompound A falls under the moderate class as determined by the BCSpermeability classification system.

Monolayer TEER results as well as post-experimental lucifer yellow fluxresults indicated the presence of functional cell monolayers throughoutthe assay. The positive control for P-gp transport, digoxin, showedpolarized transport (polarization ratio of 5.4, Table 7) indicating aproperly functioning test system.

Mass balance and non-specific binding determinations showed thatCompound A was bound to the plastic plate and/or the cells to a degreethat might have reduced the amount of compound available or diffusionand transport. As a result, the apparent P_(app) values and resultingpolarization ratios (Table 8) may underestimate the permeability ofCompound A and should be used with caution when assigning it to a BCSpermeability class as shown in Table 10.

TABLE 10 Permeability Absorption in Papp Class^([3]) Humans^([4])(cm/sec) low  <50% ≦mannitol moderate 50-89%  >mannitol and <propranololhigh ≧90% ≧propranolol

The A-B permeability observed Compound A were between those observed formannitol and propranolol. Based on the BCS permeability classification(Table 10), Compound A would be considered to be a moderate permeabilitycompound.

Compound A showed polarization ratios of 0.1-0.2, indicating that A-Bpermeability exceeded B-A permeability for this compound. This findingis inconsistent with P-gp mediated B-A efflux, and may suggestinvolvement of active A-B flux by a transporter other than P-gp.

The foregoing studies demonstrate that representative compounds of theNDGA derivatives of the present invention would be useful pharmaceuticalcompounds.

It will be appreciated by those skilled in the art that changes could bemade to the embodiments described above without departing from the broadinventive concept thereof. It is understood, therefore, that thisinvention is not limited to the particular embodiments disclosed, but itis intended to cover modifications within the spirit and scope of thepresent invention as defined by the appended claims.

REFERENCES

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We claim:
 1. A method of treating disease in a patient in need thereofcomprising the administration of an effective amount of anordihydroguaiaretic acid (NDGA) derivative compound having thefollowing general structure (Formula IV), and its pharmaceuticallyacceptable salts:

wherein X is selected from the group consisting of: -A-R; —(CH₂)_(x)Hal,where x is an integer of 1 to 10, and Hal is a halogen atom, and—(CH₂CH₂O)_(y)H, where y is an integer of 1 to 10; and acarbamate-bonded group selected from the group consisting of:

where n is an integer of 1 to 6, Z₁ is a saturated linear hydrocarbonchain of 2-6 carbons and optionally 1-3 halogen atoms, Z₂ is a 5- to7-member ring optionally containing 0-3 double bonds and optionallycontaining 1-3 atoms of any of O, N and S, and Z₃ is methyl or ethyl;wherein when X is -A-R, R is an end group and A is a linear saturatedhydrocarbon side chain bonded at one end to the respective hydroxyresidue O groups by an ether bond or a carbamate bond and at the otherend to a carbon or a heteroatom in the end group R; wherein the sidechain A is selected from the group consisting of a C₂-C₁₆ linearsaturated hydrocarbon chain optionally with 1-5 heteroatoms selectedfrom the group consisting of O, N and S, bonded to the respectivehydroxy residue O groups of NGDA through an ether bond; and 1-5 units ofa polyethylene glycol (PEG) chain; wherein R is selected from the groupconsisting of: a 5 to 7 member carbocyclic ring selected from the groupconsisting of a fully saturated ring with 1 to 3 N, O or S heteroatoms;a ring containing 1 to 3 double bonds with 1 to 3 N, O or S heteroatoms;a ring containing a carbamate bond, a urea bond, a carbonate bond or anamide bond; and a water soluble group selected from the group consistingof an alkali metal salt of sulfonic acid; an alkali metal salt ofphosphonic acid; a pharmaceutically acceptable salt; a sugar and apolyhydroxy group; and wherein when X is

a is an integer of 3 to 16 and b is an integer of 4 to
 16. 2. The methodof claim 1 wherein the composition comprises the NDGA derivative ofclaim 1 and a pharmaceutically acceptable carrier, optionally with otherpharmaceutically acceptable excipients.
 3. The method of claim 2 whereincompound is1,4-bis{3,4-bis[4-(N-piperidino)butoxyl]phenyl}-2,3-dimethyl-(2R,3S)-butane;1,4-bis{3,4-bis(2-methyl-thiazol-4-yl-methoxy)phenyl}-2,3-dimethyl-(2R,3S)-butane;1,4-bis{3,4-bis(2-(N,N′-dimethylamino)-ethoxy)phenyl}-2,3-dimethyl-(2R,3S)-butane;1,4-bis{3,4-bis(2-(N,N′-dimethylamino)-ethoxy)phenyl}-2,3-dimethyl-(2R,3S)-butane;1,4-bis{3,4-bis(2-hydroxyethoxy)phenyl}-2,3-dimethyl-(2R,3S)-butane;1,4-bis{3,4-bis[2-(2-hydroxyethoxy)ethoxyl]phenyl}-2,3-dimethyl-(2R,3S)-butane;1,4-bis[3,4-bis(2-fluoro-ethoxyl)phenyl]-2,3-dimethyl-(2R,3S)-butane;1,4-bis[3,4-bis(2-fluoro-ethoxyl)phenyl]-2,3-dimethyl-(2R,3S)-butane;1,4-bis{(3,4-bis[4-(N-morpholino)butoxyl]phenyl}-2,3-dimethyl-(2R,3S)-butane;1,4-bis{3,4-bis[4-(N-methyl-piperazino-N′-yl)butoxyl]phenyl}-2,3-dimethyl-(2R,3S)-butane;1,4-bis{3,4-bis[2-(1-methyl-piperazin-4-yl)-ethoxy]phenyl}-2,3-dimethyl-(2R,3S)-butane;1,4-bis{3,4-bis[2-(piperidin-1-yl)ethylcarbamoyloxy]phenyl}-2,3-dimethyl-(2R,3S)-butane;1,4-bis{3,4-bis[2-(morpholin-1-yl)ethylcarbamoyloxy]phenyl}-2,3-dimethyl-(2R,3S)-butane;1,4-bis{3,4-bis[(2-N,N-dimethylaminoethyl)carbamoyloxy]phenyl}-2,3-dimethyl-(2R,3S)-butane;1,4-bis{3,4-bis[(furan-2-yl)methyl-carbamoyloxy]phenyl}-2,3-dimethyl-(2R,3S)-butane;or a pharmaceutically acceptable salt thereof.
 4. The method of claim 3wherein the disease is selected from a viral infection, a proliferativedisease, an inflammatory disease, a metabolic disease, or a vasculardisease.
 5. The method of claim 4 wherein the compound is1,4-bis{3,4-bis[3-(piperidin-1yl)propoxy]phenyl}-2,3-dimethyl-(2R,3S)-butaneor a pharmaceutically acceptable salt thereof.
 6. The method of claim 4wherein the compound is1,4-bis{3,4-bis[4-(N-piperidino)butoxyl]phenyl}-2,3-dimethyl-(2R,3S)-butaneor a pharmaceutically acceptable salt thereof.
 7. The method of claim 4wherein the compound is1,4-bis{3,4-bis(2-methyl-thiazol-4-yl-methoxy)phenyl}-2,3-dimethyl-(2R,3S)-butaneor a pharmaceutically acceptable salt thereof.
 8. The method of claim 4wherein the compound is1,4-bis{3,4-bis(2-(N,N′-dimethylamino)-ethoxy)phenyl}-2,3-dimethyl-(2R,3S)-butaneor a pharmaceutically acceptable salt thereof.
 9. The method of claim 4wherein the compound is1,4-bis{3,4-bis(2-(N,N′-dimethylamino)-ethoxy)phenyl}-2,3-dimethyl-(2R,3S)-butaneor a pharmaceutically acceptable salt thereof.
 10. The method of claim 4wherein the compound is1,4-bis{3,4-bis(2-hydroxyethoxy)phenyl})-2,3-dimethyl-(2R,3S)-butane ora pharmaceutically acceptable salt thereof.
 11. The method of claim 4wherein the compound is1,4-bis{3,4-bis[2-(2-hydroxyethoxy)ethoxyl]phenyl}-2,3-dimethyl-(2R,3S)-butaneor a pharmaceutically acceptable salt thereof.
 12. The method of claim 4wherein the compound is1,4-bis[3,4-bis(2-fluoro-ethoxyl)phenyl]-2,3-dimethyl-(2R,3S)-butane ora pharmaceutically acceptable salt thereof.
 13. The method of claim 4wherein the compound is1,4-bis[3,4-bis(2-fluoro-ethoxyl)phenyl]-2,3-dimethyl-(2R,3S)-butane ora pharmaceutically acceptable salt thereof.
 14. The method of claim 4wherein the compound is1,4-bis{3,4-bis[4-(N-morpholino)butoxyl]phenyl}-2,3-dimethyl-(2R,3S)-butaneor a pharmaceutically acceptable salt thereof.
 15. The method of claim 4wherein the compound is1,4-bis{3,4-bis[4-(N-methyl-piperazino-N′-yl)butoxyl]phenyl}-2,3-dimethyl-(2R,3S)-butaneor a pharmaceutically acceptable salt thereof.
 16. The method of claim 1wherein the compound is1,4-bis{3,4-bis[2-(1-methyl-piperazin-4-yl)-ethoxy]phenyl}-2,3-dimethyl-(2R,3S)-butaneor a pharmaceutically acceptable salt thereof.
 17. The method of claim 1wherein the compound is1,4-bis{3,4-bis[2-(piperidin-1-yl)ethylcarbamoyloxy]phenyl}-2,3-dimethyl-(2R,3S)-butaneor a pharmaceutically acceptable salt thereof.
 18. The method of claim 1wherein the compound is1,4-bis{3,4-bis[2-(morpholin-1-yl)ethylcarbamoyloxy]phenyl}-2,3-dimethyl-(2R,3S)-butaneor a pharmaceutically acceptable salt thereof.
 19. The method of claim 1wherein the compound is1,4-bis{3,4-bis[(2-N,N-dimethylaminoethyl)carbamoyloxy]phenyl}-2,3-dimethyl-(2R,3S)-butaneor a pharmaceutically acceptable salt thereof.
 20. The method of claim 1wherein the compound is1,4-bis{3,4-bis[(furan-2-yl)methyl-carbamoyloxy]phenyl}-2,3-dimethyl-(2R,3S)-butaneor a pharmaceutically acceptable salt thereof.